PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

PubMed

Nishizaki, Tomoyuki

2018-01-01

In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Bed-rest studies: Fluid and electrolyte responses

NASA Technical Reports Server (NTRS)

Greenleaf, J. E.

1983-01-01

Confinement in the horizontal position for 2 to 3 weeks results in a chronic decrease in plasma volume, increased interstitial fluid volume, and unchanged or slightly increased extracellular fluid volume. Concentrations of blood electrolytes, glucose, and nitrogenous constituents remain within normal limits of variability when maintenance levels of isometric or isotonic exercise are performed for 1 hr/day. Hematocrit and plasma osmolality can be elevated significantly throughout bed rest (BR). Significant diuresis occurs on the first day, and increases in urine Na and Ca continue throughout BR, although voluntary fluid intake is unchanged. Urine Na and K are evaluated during the second week of BR in spite of stabilization of PV and extracellular volume. The initial diuresis probably arises from the extracellular fluid while subsequent urine loss above control levels must come from the intracellular fluid. Preservation of the extracellular volume takes precedance over maintenance of the intracellular fluid volume. The functioning of a natriuretic factor (hormone) to account for the continued increased loss of Na in the urine is suggested.

Training-induced elevations in extracellular lactate in hippocampus and striatum: Dissociations by cognitive strategy and type of reward.

PubMed

Newman, Lori A; Scavuzzo, Claire J; Gold, Paul E; Korol, Donna L

2017-01-01

Recent evidence suggests that astrocytes convert glucose to lactate, which is released from the astrocytes and supports learning and memory. This report takes a multiple memory perspective to test the role of astrocytes in cognition using real-time lactate measurements during learning and memory. Extracellular lactate levels in the hippocampus or striatum were determined with lactate biosensors while rats were learning place (hippocampus-sensitive) or response (striatum-sensitive) versions of T-mazes. In the first experiment, rats were trained on the place and response tasks to locate a food reward. Extracellular lactate levels in the hippocampus increased beyond those of feeding controls during place training but not during response training. However, striatal lactate levels did not increase beyond those of controls when rats were trained on either the place or the response version of the maze. Because food ingestion itself increased blood glucose and brain lactate levels, the contribution of feeding may have confounded the brain lactate measures. Therefore, we conducted a second similar experiment using water as the reward. A very different pattern of lactate responses to training emerged when water was used as the task reward. First, provision of water itself did not result in large increases in either brain or blood lactate levels. Moreover, extracellular lactate levels increased in the striatum during response but not place learning, whereas extracellular lactate levels in the hippocampus did not differ across tasks. The findings from the two experiments suggest that the relative engagement of the hippocampus and striatum dissociates not only by task but also by reward type. The divergent lactate responses of the hippocampus and striatum in place and response tasks under different reward conditions may reflect ethological constraints tied to foraging for food and water. Copyright © 2016 Elsevier Inc. All rights reserved.

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

PubMed

Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

2007-10-01

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Effects of chronic treatment with escitalopram or citalopram on extracellular 5-HT in the prefrontal cortex of rats: role of 5-HT1A receptors

PubMed Central

Ceglia, I; Acconcia, S; Fracasso, C; Colovic, M; Caccia, S; Invernizzi, R W

2004-01-01

Microdialysis was used to study the acute and chronic effects of escitalopram (S-citalopram; ESCIT) and chronic citalopram (CIT), together with the 5-HT1A receptor antagonist WAY100,635 (N-[2-[methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride) and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on extracellular 5-hydroxytryptamine (5-HT) levels in the rat prefrontal cortex. Extracellular 5-HT rose to 234 and 298% of basal values after subcutaneous (s.c.) acute doses of 0.15 and 0.63 mg kg−1 ESCIT. No further increase was observed at 2.5 mg kg−1 ESCIT (290%). The effect of 13-day s.c. infusion of 10 mg kg−1day−1 ESCIT on extracellular 5-HT (422% of baseline) was greater than after 2 days (257% of baseline), whereas exposure to ESCIT was similar. In contrast, the increase in extracellular 5-HT induced by the infusion of CIT for 2 (306%) and 13 days (302%) was similar. However, brain and plasma levels of S-citalopram in rats infused with CIT for 13 days were lower than after 2 days. Acute treatment with 2.5 mg kg−1 ESCIT or 5 mg kg−1 CIT raised extracellular 5-HT by 243 and 276%, respectively, in rats given chronic vehicle but had no effect in rats given ESCIT (10 mg kg−1 day−1) or CIT (20 mg kg−1 day−1) for 2 or 13 days, suggesting that the infused doses had maximally increased extracellular 5-HT. WAY100,635 (0.1 mg kg−1 s.c.) increased extracellular 5-HT levels by 168, 174 and 169% of prechallenge values in rats infused with vehicle or ESCIT for 2 or 13 days, respectively. WAY100,635 enhanced extracellular 5-HT levels to 226, 153 and 164% of prechallenge values in rats infused with vehicle or CIT for 2 and 13 days, respectively. 8-OH-DPAT (0.025 mg kg−1) reduced extracellular 5-HT by 54% in control rats, but had no effect in those given ESCIT and CIT for 13 days. This series of experiments led to the conclusion that chronic treatment with ESCIT desensitizes the 5-HT1A receptors, regulating the release of 5-HT in the prefrontal cortex and enhances the effect of the drug on extracellular 5-HT. They also indicate that chronic treatment with ESCIT and CIT did not prevent WAY100,635 from raising extracellular 5-HT. PMID:15148253

Extracellular metabolites in the cortex and hippocampus of epileptic patients.

PubMed

Cavus, Idil; Kasoff, Willard S; Cassaday, Michael P; Jacob, Ralph; Gueorguieva, Ralitza; Sherwin, Robert S; Krystal, John H; Spencer, Dennis D; Abi-Saab, Walid M

2005-02-01

Interictal brain energy metabolism and glutamate-glutamine cycling are impaired in epilepsy and may contribute to seizure generation. We used the zero-flow microdialysis method to measure the extracellular levels of glutamate, glutamine, and the major energy substrates glucose and lactate in the epileptogenic and the nonepileptogenic cortex and hippocampus of 38 awake epileptic patients during the interictal period. Depth electrodes attached to microdialysis probes were used to identify the epileptogenic and the nonepileptogenic sites. The epileptogenic hippocampus had surprisingly high basal glutamate levels, low glutamine/glutamate ratio, high lactate levels, and indication for poor glucose utilization. The epileptogenic cortex had only marginally increased glutamate levels. We propose that interictal energetic deficiency in the epileptogenic hippocampus could contribute to impaired glutamate reuptake and glutamate-glutamine cycling, resulting in persistently increased extracellular glutamate, glial and neuronal toxicity, increased lactate production together with poor lactate and glucose utilization, and ultimately worsening energy metabolism. Our data suggest that a different neurometabolic process underlies the neocortical epilepsies.

Dopamine D1 receptor-dependent regulation of extracellular citrulline level in the rat nucleus accumbens during conditioned fear response.

PubMed

Saulskaya, Natalia B; Fofonova, Nellia V; Sudorghina, Polina V; Saveliev, Sergey A

2008-08-01

Nucleus accumbens (N.Acc) contains a subclass of nitric oxide (NO)-generating interneurons that are presumably regulated by the dopamine input. Receptor mechanisms underlying dopamine-NO interaction in the N.Acc are poorly understood. In the current study, we used in vivo microdialysis combined with high-performance liquid chromatography to examine participation of dopamine D1 receptors in regulation of extracellular levels of citrulline (an NO co-product) in the medial N.Acc of Sprague-Dawley rats during both pharmacological challenge and a conditioned fear response. The intraaccumbal infusion of the D1 receptor agonist SKF-38393 (100-500 microM) increased dose-dependently the local dialysate citrulline levels. The SKF-38393-induced increase in extracellular citrulline was prevented by intraaccumbal infusions of 500 microM 7-nitroindazole, a neuronal NO synthase inhibitor. In behavioral microdialysis experiment, the accumbal levels of extracellular citrulline markedly increased in rats given a mild footshock paired with tone. The presentation of the tone previously paired with footshock (the conditioned fear response) produced a "conditioned" rise of extracellular citrulline levels in the N.Acc which was attenuated by intraaccumbal infusion of 100 microM SCH-23390, a dopamine D1 receptor antagonist, and prevented by intraaccumbal infusion of 500 microM 7-nitroindazole. The results suggest that in the N.Acc, the dopamine D1 receptors might regulate the neuronal NO synthase activity; this dopamine-dependent mechanism seems to participate in activation of the neuronal NO synthase and probably NO formation in this brain area during the conditioned fear response.

Bed-rest studies - Fluid and electrolyte responses

NASA Technical Reports Server (NTRS)

Greenleaf, J. E.

1983-01-01

Confinement in the horizontal position for 2 to 3 weeks results in a chronic decrease in plasma volume, increased interstitial fluid volume, and unchanged or slightly increased extracellular fluid volume. Concentrations of blood electrolytes, glucose, and nitrogenous constituents remain within normal limits of variability when maintenance levels of isometric or isotonic exercise are performed for 1 hr/day. Hematocrit and plasma osmolality can be elevated significantly throughout bed rest (BR). Significant diuresis occurs on the first day, and increases in urine Na and Ca continue throughout BR, although voluntary fluid intake is unchanged. Urine Na and K are evaluated during the second week of BR in spite of stabilization of PV and extracellular volume. The initial diuresis probably arises from extracellular fluid while subsequent urine loss above control levels must come from the intracellular fluid. Preservation of the extracellular volume takes precedance over maintenance of the intracellular fluid volume. The functioning of a natriuretic factor (hormone) to account for the continued increased loss of Na in the urine is suggested. Previously announced in STAR as N83-24160

Nucleosomes and neutrophil activation in sickle cell disease painful crisis

PubMed Central

Schimmel, Marein; Nur, Erfan; Biemond, Bart J.; van Mierlo, Gerard J.; Solati, Shabnam; Brandjes, Dees P.; Otten, Hans-Martin; Schnog, John-John; Zeerleder, Sacha

2013-01-01

Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ0-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ+-thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome. PMID:23911704

Anethole dithiolethione, a putative neuroprotectant, increases intracellular and extracellular glutathione levels during starvation of cultured astroglial cells.

PubMed

Dringen, R; Hamprecht, B; Drukarch, B

1998-12-01

Astroglial cells protect neurons against oxidative damage. The antioxidant glutathione plays a pivotal role in the neuroprotective action of astroglial cells which is impaired following loss of glutathione. Anethole dithiolethione (ADT), a sulfur-containing compound which is used in humans as a secretagogue, increases glutathione levels in cultured astroglial cells under "physiological" conditions and is thought thereby to protect against oxidative damage. Presently, we report the effect of ADT (3-100 microM) on glutathione content of and efflux from rat primary astroglia-rich cultures under "pathological" conditions, i.e., extended deprivation of glucose and amino acids. Although cellular viability was not affected significantly, starvation of these cultures for 24 h in a bicarbonate buffer lacking glucose and amino acids led to a decrease in glutathione and protein content of approximately 43% and 40%, respectively. Although no effect on the protein loss occurred, the presence of ADT during starvation counteracted the starvation-induced loss of intracellular glutathione in a concentration-dependent way. At a concentration of 100 microM ADT even a significant increase in astroglial glutathione content was noted after 24 h of starvation. Alike intracellular glutathione levels, the amount of glutathione found in the buffer was elevated substantially if ADT was present during starvation. This ADT-mediated, apparent increase in glutathione efflux was additive to the stimulatory effect on extracellular glutathione levels of acivicin (100 microM), an inhibitor of extracellular enzymatic glutathione breakdown. However, the ADT-induced elevation of both intra- and extracellular glutathione content during starvation was prevented completely by coincubation with buthionine sulfoximine (10 microM), an inhibitor of glutathione synthesis. These results demonstrate that, most likely through stimulation of glutathione synthesis, ADT enables astroglial cells to maintain higher intra- and extracellular levels of glutathione under adverse conditions. Considering the lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of the compound is warranted.

Spatial distribution and expression of intracellular and extracellular acid phosphatases of cluster roots at different developmental stages in white lupin.

PubMed

Tang, Hongliang; Li, Xiaoqing; Zu, Chao; Zhang, Fusuo; Shen, Jianbo

2013-09-15

Acid phosphatases (APases) play a key role in phosphorus (P) acquisition and recycling in plants. White lupin (Lupinus albus L.) forms cluster roots (CRs) and produces large amounts of APases under P deficiency. However, the relationships between the activity of intracellular and extracellular APases (EC 3.1.3.2) and CR development are not fully understood. Here, comparative studies were conducted to examine the spatial variation pattern of APase activity during CR development using the enzyme-labelled fluorescence-97 (ELF-97) and the p-nitrophenyl phosphate methods. The activity of intracellular and extracellular APases was significantly enhanced under P deficiency in the non-CRs and CRs at different developmental stages. These two APases exhibited different spatial distribution patterns during CR development, and these distribution patterns were highly modified by P deficiency. The activity of extracellular APase increased steadily with CR development from meristematic, juvenile, mature to senescent stages under P deficiency. In comparison, P deficiency-induced increase in the activity of intracellular APase remained relatively constant during CR development. Increased activity of intracellular and extracellular APases was associated with enhanced expression of LaSAP1 encoding intracellular APase and LaSAP2 encoding extracellular APase. The expression levels of these two genes were significantly higher at transcriptional level in both mature and senescent CRs. Taken together, these findings demonstrate that both activity and gene expression of intracellular or extracellular APases exhibit a differential response pattern during CR development, depending on root types, CR developmental stages and P supply. Simultaneous in situ determination of intracellular and extracellular APase activity has proved to be an effective approach for studying spatial variation of APases during CR development. Copyright © 2013 Elsevier GmbH. All rights reserved.

β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons.

PubMed

Hossain, Murad; Wickramasekara, Rochelle N; Carvelli, Lucia

2014-07-01

β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. Copyright © 2013 Elsevier Ltd. All rights reserved.

β-phenylethylamine Requires the Dopamine Transporter to Increase Extracellular Dopamine in C. elegans Dopaminergic Neurons

PubMed Central

Hossain, Murad; Wickramasekara, Rochelle N.; Carvelli, Lucia

2013-01-01

β-phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. PMID:24161617

Cell swelling increases the severity of spreading depression in Locusta migratoria

PubMed Central

Chin, Brittany; Witiuk, Kelsey L. M.

2015-01-01

Progressive accumulation of extracellular potassium ions can trigger propagating waves of spreading depression (SD), which are associated with dramatic increases in extracellular potassium levels ([K+]o) and arrest in neural activity. In the central nervous system the restricted nature of the extracellular compartment creates an environment that is vulnerable to disturbances in ionic homeostasis. Here we investigate how changes in the size of the extracellular space induced by alterations in extracellular osmolarity affect locust SD. We found that hypotonic exposure increased susceptibility to experimentally induced SD evidenced by a decrease in the latency to onset and period between individual events. Hypertonic exposure was observed to delay the onset of SD or prevent the occurrence altogether. Additionally, the magnitude of extracellular K+ concentration ([K+]o) disturbance during individual SD events was significantly greater and they were observed to propagate more quickly under hypotonic conditions compared with hypertonic conditions. Our results are consistent with a conclusion that hypotonic exposure reduced the size of the extracellular compartment by causing cell swelling and thus facilitated the accumulation of K+ ions. Lastly, we found that pharmacologically reducing the accumulation of extracellular K+ using the K+ channel blocker tetraethylammonium slowed the rate of SD propagation while increasing [K+]o through inhibition of the Na-K-2Cl cotransporter increased propagation rates. Overall our findings indicate that treatments or conditions that act to reduce the accumulation of extracellular K+ help to protect against the development of SD and attenuate the spread of ionic disturbance adding to the evidence that diffusion of K+ is a leading event during locust SD. PMID:26378209

Dopamine physiology in the basal ganglia of male zebra finches during social stimulation.

PubMed

Ihle, Eva C; van der Hart, Marieke; Jongsma, Minke; Tecott, Larry H; Doupe, Allison J

2015-06-01

Accumulating evidence suggests that dopamine (DA) is involved in altering neural activity and gene expression in a zebra finch cortical-basal ganglia circuit specialized for singing, upon the shift between solitary singing and singing as a part of courtship. Our objective here was to sample changes in the extracellular concentrations of DA in Area X of adult and juvenile birds, to test the hypothesis that DA levels would change similarly during presentation of a socially salient stimulus in both age groups. We used microdialysis to sample the extracellular milieu of Area X in awake, behaving adult and juvenile male zebra finches, and analysed the dialysate using high-performance liquid chromatography coupled with electrochemical detection. The extracellular levels of DA in Area X increased significantly during both female presentation to adult males and tutor presentation to juvenile males. DA levels were not correlated with the time spent singing. We also reverse-dialysed Area X with pharmacologic agents that act either on DA systems directly or on norepinephrine, and found that all of these agents significantly increased DA levels (3- to 10-fold) in Area X. These findings suggest that changes in extracellular DA levels can be stimulated similarly by very different social contexts (courtship and interaction with tutor), and influenced potently by dopaminergic and noradrenergic drugs. These results raise the possibility that the arousal level or attentional state of the subject (rather than singing behavior) is the common feature eliciting changes in extracellular DA concentration. © 2015 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Effects of extracellular modulation through hypoxia on the glucose metabolism of human breast cancer stem cells

NASA Astrophysics Data System (ADS)

Yustisia, I.; Jusman, S. W. A.; Wanandi, S. I.

2017-08-01

Cancer stem cells have been reported to maintain stemness under certain extracellular changes. This study aimed to analyze the effect of extracellular O2 level modulation on the glucose metabolism of human CD24-/CD44+ breast cancer stem cells (BCSCs). The primary BCSCs (CD24-/CD44+ cells) were cultured under hypoxia (1% O2) for 0.5, 4, 6, 24 and 48 hours. After each incubation period, HIF1α, GLUT1 and CA9 expressions, as well as glucose metabolism status, including glucose consumption, lactate production, O2 consumption and extracellular pH (pHe) were analyzed using qRT-PCR, colorimetry, fluorometry, and enzymatic reactions, respectively. Hypoxia caused an increase in HIF1α mRNA expressions and protein levels and shifted the metabolic states to anaerobic glycolysis, as demonstrated by increased glucose consumption and lactate production, as well as decreased O2 consumption and pHe. Furthermore, we demonstrated that GLUT1 and CA9 mRNA expressions simultaneously increased, in line with HIF1α expression. In conclusion, modulation of the extracellular environment of human BCSCs through hypoxia shifedt the metabolic state of BCSCs to anaerobic glycolysis, which might be associated with GLUT1 and CA9 expressions regulated by HIFlα transcription factor.

Hepatocyte growth factor: a regulator of extracellular matrix genes in mouse mesangial cells.

PubMed

Laping, N J; Olson, B A; Ho, T; Ziyadeh, F N; Albrightson, C R

2000-04-01

The potential role of hepatocyte growth factor (HGF) in regulating extracellular matrix in mouse mesangial cells (MMC) was evaluated. Functional HGF receptors were deed in MMC by HGF-induced extracellular acidification, a response that was inhibited by the HGF inhibitor HGF/NK2, a splice variant expressing the N-terminal domain through the second kringle domain HGF also increased fibronectin and collagen alpha1 (IV) mRNA levels in these cells; the increases were associated with a concentration-dependent increase in transcriptional activity of the collagen alpha1 (IV) gene. HGF also stimulated fibronectin and collagen alpha1 (IV) mRNA levels in primary rabbit proximal tubule epithelial cells To evaluate the potential consequences of chronic elevation of HGF on renal fuction, HGF was administered continuously for 18 days to normal and diabetic C57BLKS/J lepr(db) mice. In the diabetic mice, HGF reduced creatinine clearance and increased microalbuminuria, indicating that chronic exposure to HGF impairs renal function. Thus, chronically elevated HGF may contribute to the progression of chronic renal disease in diabetes by decreasing the glomerular filtration rate and possibly promoting the accumulation of extracellular matrix.

Rapid fluctuations in extracellular brain glucose levels induced by natural arousing stimuli and intravenous cocaine: fueling the brain during neural activation

PubMed Central

Lenoir, Magalie

2012-01-01

Glucose, a primary energetic substrate for neural activity, is continuously influenced by two opposing forces that tend to either decrease its extracellular levels due to enhanced utilization in neural cells or increase its levels due to entry from peripheral circulation via enhanced cerebral blood flow. How this balance is maintained under physiological conditions and changed during neural activation remains unclear. To clarify this issue, enzyme-based glucose sensors coupled with high-speed amperometry were used in freely moving rats to evaluate fluctuations in extracellular glucose levels induced by brief audio stimulus, tail pinch (TP), social interaction with another rat (SI), and intravenous cocaine (1 mg/kg). Measurements were performed in nucleus accumbens (NAcc) and substantia nigra pars reticulata (SNr), which drastically differ in neuronal activity. In NAcc, where most cells are powerfully excited after salient stimulation, glucose levels rapidly (latency 2–6 s) increased (30–70 μM or 6–14% over baseline) by all stimuli; the increase differed in magnitude and duration for each stimulus. In SNr, where most cells are transiently inhibited by salient stimuli, TP, SI, and cocaine induced a biphasic glucose response, with the initial decrease (−20–40 μM or 5–10% below baseline) followed by a reboundlike increase. The critical role of neuronal activity in mediating the initial glucose response was confirmed by monitoring glucose currents after local microinjections of glutamate (GLU) or procaine (PRO). While intra-NAcc injection of GLU transiently increased glucose levels in this structure, intra-SNr PRO injection resulted in rapid, transient decreases in SNr glucose. Therefore, extracellular glucose levels in the brain change very rapidly after physiological and pharmacological stimulation, the response is structure specific, and the pattern of neuronal activity appears to be a critical factor determining direction and magnitude of physiological fluctuations in glucose levels. PMID:22723672

Nonvesicular Release of Glutamate by Glial xCT Transporters Suppresses Glutamate Receptor Clustering In Vivo

PubMed Central

Augustin, Hrvoje; Grosjean, Yael; Chen, Kaiyun; Sheng, Qi; Featherstone, David E.

2008-01-01

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named “genderblind” (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200–300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses. PMID:17202478

[Mediolateral gradient of the nucleus accumbens nitrergic activation during exploratory behavior].

PubMed

Saul'skaia, N B; Sudorgina, P V

2012-04-01

In Sprague-Dawley rats, by means of in vivo microdialysis combined with HPLC analysis it has been shown that an exploratory behavior in a new environment is accompanied by a rise in extracellular levels of citrulline (an NO co-product) in the mediolateral regions of the n. accumbens with the maximum observed in the medial n. accumbens. Infusions of 7-nitroindazole (0.5 mM), a neuronal NO synthase inhibitor, into the medial n. accumbens prevented the exploration-induced rise of extracellular citrulline levels in this area. The second presentation of the same chamber did not produce any significant changes of extracellular citrulline levels in the medial n. accumbens, although there was a tendency of a small increase. The presentation of a familiar chamber did not affect citrulline extracellular levels in this area. The data obtained indicate for the first time that exploratory activity in a new environment is accompanied by the nitrergic activation in the entire n. accumbens with the maximal activation in the medial part of this brain area.

The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

PubMed

Gołembiowska, Krystyna; Dziubina, Anna

2012-08-01

It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson's disease (PD). In our previous study, we demonstrated that adenosine A(2A) receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A(2A) receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. L-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A(2A) receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage mechanism in early PD.

Inhibition of prolyl oligopeptidase by KYP-2047 fails to increase the extracellular neurotensin and substance P levels in rat striatum.

PubMed

Jalkanen, Aaro J; Savolainen, Katja; Forsberg, Markus M

2011-09-15

Prolyl oligopeptidase (PREP, EC 3.4.21.26) hydrolyzes neuropeptides, such as neurotensin and substance P in vitro, but its importance in the in vivo metabolism of these peptides has not been proved. This is the first report where intracerebral microdialysis combined with highly sensitive radioimmunoassay has been used to investigate the effect of PREP inhibition on the brain extracellular peptide levels in conscious rats. We show that PREP inhibition by KYP-2047 (50μmol/kg=17mg/kg, intraperitoneally, that effectively inhibits PREP in the brain), has no effect on the neurotensin and substance P levels in the striatum extracellular space. This provides a further piece of evidence in support of the proposition that PREP is not significantly responsible for the in vivo cleavage of substance P or neurotensin, and that occasional positive cognitive effects associated with some PREP inhibitors are not mediated through elevated extracellular levels of these peptides. Direct regulation of peptide processing by PREP is not likely because the enzyme is located intracellularly and the peptide substrates are mostly extracellular. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Response of extracellular zinc in the ventral hippocampus against novelty stress.

PubMed

Takeda, Atsushi; Sakurada, Naomi; Kanno, Shingo; Minami, Akira; Oku, Naoto

2006-10-01

An extensive neuronal activity takes place in the hippocampus during exploratory behavior. However, the role of hippocampal zinc in exploratory behavior is poorly understood. To analyze the response of extracellular zinc in the hippocampus against novelty stress, rats were placed for 50 min in a novel environment once a day for 8 days. Extracellular glutamate in the hippocampus was increased during exploratory behavior on day 1, whereas extracellular zinc was decreased. The same phenomenon was observed during exploratory behavior on day 2 and extracellular zinc had returned to the basal level during exploratory behavior on day 8. To examine the significance of the decrease in extracellular zinc in exploratory activity, exploratory behavior was observed during perfusion with 1 mm CaEDTA, a membrane-impermeable zinc chelator. Locomotor activity in the novel environment was decreased by perfusion with CaEDTA. The decrease in extracellular zinc and the increase in extracellular glutamate in exploratory period were abolished by perfusion with CaEDTA. These results suggest that zinc uptake by hippocampal cells is linked to exploratory activity and is required for the activation of the glutamatergic neurotransmitter system. The zinc uptake may be involved in the response to painless psychological stress or in the cognitive processes.

Osteogenic differentiation capacity of human mesenchymal stromal cells in response to extracellular calcium with special regard to connexin 43.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Wolf, Verena; Fietz, Daniela; Mazurek, Sybille; Hanke, Thomas; Moritz, Andreas; Arnhold, Stefan; Wenisch, Sabine

2017-01-01

The effects of extracellular calcium on osteogenic differentiation capacity of human bone-derived mesenchymal stromal cells with special regard to connexin 43 (cx43) have been investigated by means of cell culture experiments. Mesenchymal stromal cells isolated from human cancellous bone were cultured on tissue culture plates at different calcium ion (Ca 2+ ) concentrations (1.8mmoll -1 , 10mmoll -1 , 20mmoll -1 ). Cell responses were evaluated by quantitative RT-PCR, immunofluorescence staining, and Lucifer Yellow fluorescence uptake experiments. It could be shown that increasing Ca 2+ concentrations correlate with increasing cx43 and bone sialoprotein mRNA levels as well as with enhanced cx43 fluorescence signaling and matrix mineralization of the cultures as shown by von Kossa staining. Hemichannel gating - assessed by Lucifer Yellow uptake - increases with increasing extracellular Ca 2+ concentrations suggesting that regulatory effects at the hemichannel level are calcium-dependent. Copyright © 2016 Elsevier GmbH. All rights reserved.

Transcranial direct-current stimulation increases extracellular dopamine levels in the rat striatum

PubMed Central

Tanaka, Tomoko; Takano, Yuji; Tanaka, Satoshi; Hironaka, Naoyuki; Kobayashi, Kazuto; Hanakawa, Takashi; Watanabe, Katsumi; Honda, Manabu

2013-01-01

Background: Transcranial direct-current stimulation (tDCS) is a non-invasive procedure that achieves polarity-dependent modulation of neuronal membrane potentials. It has recently been used as a functional intervention technique for the treatment of psychiatric and neurological diseases; however, its neuronal mechanisms have not been fully investigated in vivo. Objective/Hypothesis: To investigate whether the application of cathodal or anodal tDCS affects extracellular dopamine and serotonin levels in the rat striatum. Methods: Stimulation and in vivo microdialysis were carried out under urethane anesthesia, and microdialysis probes were slowly inserted into the striatum. After the collection of baseline fractions in the rat striatum, cathodal or anodal tDCS was applied continuously for 10 min with a current intensity of 800 μA from an electrode placed on the skin of the scalp. Dialysis samples were collected every 10 min until at least 400 min after the onset of stimulation. Results: Following the application of cathodal, but not anodal, tDCS for 10 min, extracellular dopamine levels increased for more than 400 min in the striatum. There were no significant changes in extracellular serotonin levels. Conclusion: These findings suggest that tDCS has a direct and/or indirect effect on the dopaminergic system in the rat basal ganglia. PMID:23596399

Proteomic Analysis Reveals Aberrant O-GlcNAcylation of Extracellular Proteins from Breast Cancer Cell Secretion.

PubMed

Netsirisawan, Pukkavadee; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

2015-01-01

O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells. Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry. O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC. O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

PubMed

Wilkins, Heather M; Koppel, Scott J; Weidling, Ian W; Roy, Nairita; Ryan, Lauren N; Stanford, John A; Swerdlow, Russell H

2016-12-01

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aβ 1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Clonazepam increases in vivo striatal extracellular glucose in diabetic rats after glucose overload.

PubMed

Gomez, Rosane; Barros, Helena M T

2003-12-01

Hyperglycemia modulates brain function, including neuronal excitability, neurotransmitter release and behavioral changes. There may be connections between the GABAergic system, glucose sensing neurons and glucose in the neuronal environment that shed light on the mechanism by which GABA(A) agents influence depressive behavior in diabetic rats submitted to the forced swimming test. We aimed to investigate whether clonazepam (CNZ), a GABA(A) receptor positive modulator, modifies in vivo striatal extracellular glucose levels in diabetic rats under fasting condition or after oral glucose overload. Streptozotocin diabetic and nondiabetic rats were submitted to in vivo striatal microdialysis. Perfusate samples were collected at baseline, during fasting and following administration of CNZ (0.25 mg/kg) and oral glucose overload. Blood glucose and striatal extracellular glucose were measured simultaneously at several time points. Fasting striatal glucose levels were higher in diabetic than in nondiabetic rats and the differences between these animals were maintained after glucose overload. The increases in extracellular striatal glucose after glucose overload were around 40% and blood to brain transference was decreased in diabetics. CNZ treatment paradoxically increased striatal glucose after glucose overload in diabetic rats, which may mark the dysfunction in brain glucose homeostasis.

Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

PubMed Central

Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

2012-01-01

Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

PubMed

Lin, Michael K; Yang, Jin; Hsu, Chun Wei; Gore, Anuradha; Bassuk, Alexander G; Brown, Lewis M; Colligan, Ryan; Sengillo, Jesse D; Mahajan, Vinit B; Tsang, Stephen H

2018-05-05

High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Tetrodotoxin-dependent glutamate release in the rat nucleus accumbens during concurrent presentation of appetitive and conditioned aversive stimuli.

PubMed

Saulskaya, Natalia B; Soloviova, Nina A

2004-12-30

In vivo microdialysis combined with a high-performance liquid chromatography was used to monitor extracellular glutamate (GLU) levels in the nucleus accumbens (N.Acc) of Sprague-Dawley rats during their behavioral responses to the concurrent presentation of appetitive and conditioned aversive stimuli. The presentation of a highly palatable diet followed by a tone previously paired with footshock to rats trained to take a pellet of the diet under these experimental conditions resulted in a marked and short lasting increase in extracellular glutamate, whereas the tone alone had no effect. A similar increase of the glutamate release was observed during the presentation of a piece of rubber instead of the diet. In both cases, the increase in extracellular glutamate was completely prevented by intra-accumbal infusions through the dialysis probe of 1 microM tetrodotoxin (a voltage-dependent Na(+) channel blocker), whereas (S)-4-carboxyphenylglycine (a cystine/glutamate exchange blocker, 5 microM) had no effect. The data obtained suggest that behavioral responses to unpredicted change in motivational value of expected reward appear to be associated with an increase of the extracellular glutamate level in the nucleus accumbens, and impulse-dependent synaptic release, rather than non-vesicular glutamate release via cystine/glutamate exchange, is responsible for this phenomenon.

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

PubMed Central

2004-01-01

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated. PMID:15324306

Rapid microelectrode measurements and the origin and regulation of extracellular glutamate in rat prefrontal cortex.

PubMed

Hascup, Erin R; Hascup, Kevin N; Stephens, Michelle; Pomerleau, Francois; Huettl, Peter; Gratton, Alain; Gerhardt, Greg A

2010-12-01

Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal versus astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (sodium channel blocker), produced a significant (∼ 40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally applied ω-conotoxin (MVIIC; ∼ 50%; calcium channel blocker), and the mGluR(2/3) agonist, LY379268 (∼ 20%), and a significant increase with the mGluR(2/3) antagonist LY341495 (∼ 40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (glutamate transporter inhibitor) produced an ∼ 120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of tetrodotoxin completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the microelectrode array technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically -evoked event is entirely neuronally derived. © 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.

Expression of intra- and extracellular granzymes in patients with typhoid fever.

PubMed

de Jong, Hanna K; Garcia-Laorden, Maria Isabel; Hoogendijk, Arie J; Parry, Christopher M; Maude, Rapeephan R; Dondorp, Arjen M; Faiz, Mohammed Abul; van der Poll, Tom; Wiersinga, Willem Joost

2017-07-01

Typhoid fever, caused by the intracellular pathogen Salmonella (S.) enterica serovar Typhi, remains a major cause of morbidity and mortality worldwide. Granzymes are serine proteases promoting cytotoxic lymphocytes mediated eradication of intracellular pathogens via the induction of cell death and which can also play a role in inflammation. We aimed to characterize the expression of extracellular and intracellular granzymes in patients with typhoid fever and whether the extracellular levels of granzyme correlated with IFN-γ release. We analyzed soluble protein levels of extracellular granzyme A and B in healthy volunteers and patients with confirmed S. Typhi infection on admission and day of discharge, and investigated whether this correlated with interferon (IFN)-γ release, a cytokine significantly expressed in typhoid fever. The intracellular expression of granzyme A, B and K in subsets of lymphocytic cells was determined using flow cytometry. Patients demonstrated a marked increase of extracellular granzyme A and B in acute phase plasma and a correlation of both granzymes with IFN-γ release. In patients, lower plasma levels of granzyme B, but not granzyme A, were found at day of discharge compared to admission, indicating an association of granzyme B with stage of disease. Peripheral blood mononuclear cells of typhoid fever patients had a higher percentage of lymphocytic cells expressing intracellular granzyme A and granzyme B, but not granzyme K, compared to controls. The marked increase observed in extra- and intracellular levels of granzyme expression in patients with typhoid fever, and the correlation with stage of disease, suggests a role for granzymes in the host response to this disease.

Expression of intra- and extracellular granzymes in patients with typhoid fever

PubMed Central

Garcia-Laorden, Maria Isabel; Hoogendijk, Arie J.; Parry, Christopher M.; Maude, Rapeephan R.; Dondorp, Arjen M.; Faiz, Mohammed Abul; van der Poll, Tom; Wiersinga, Willem Joost

2017-01-01

Background Typhoid fever, caused by the intracellular pathogen Salmonella (S.) enterica serovar Typhi, remains a major cause of morbidity and mortality worldwide. Granzymes are serine proteases promoting cytotoxic lymphocytes mediated eradication of intracellular pathogens via the induction of cell death and which can also play a role in inflammation. We aimed to characterize the expression of extracellular and intracellular granzymes in patients with typhoid fever and whether the extracellular levels of granzyme correlated with IFN-γ release. Methods and principal findings We analyzed soluble protein levels of extracellular granzyme A and B in healthy volunteers and patients with confirmed S. Typhi infection on admission and day of discharge, and investigated whether this correlated with interferon (IFN)-γ release, a cytokine significantly expressed in typhoid fever. The intracellular expression of granzyme A, B and K in subsets of lymphocytic cells was determined using flow cytometry. Patients demonstrated a marked increase of extracellular granzyme A and B in acute phase plasma and a correlation of both granzymes with IFN-γ release. In patients, lower plasma levels of granzyme B, but not granzyme A, were found at day of discharge compared to admission, indicating an association of granzyme B with stage of disease. Peripheral blood mononuclear cells of typhoid fever patients had a higher percentage of lymphocytic cells expressing intracellular granzyme A and granzyme B, but not granzyme K, compared to controls. Conclusion The marked increase observed in extra- and intracellular levels of granzyme expression in patients with typhoid fever, and the correlation with stage of disease, suggests a role for granzymes in the host response to this disease. PMID:28749963

Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p

PubMed Central

Wu, Boqian; Ottow, Kim; Poulsen, Peter; Gaber, Richard F.; Albers, Eva; Kielland-Brandt, Morten C.

2006-01-01

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand. PMID:16651382

Titin-based stiffening of muscle fibers in Ehlers-Danlos Syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottenheijm, Coen A.C.; Voermans, Nicol C.; Hudson, Bryan D.

Tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence leads to Ehlers-Danlos Syndrome (EDS). TNX-deficient EDS patients present with joint hypermobility and muscle weakness attributable to increased compliance of the extracellular matrix. We hypothesized that in response to the increased compliance of the extracellular matrix in TNX-deficient EDS patients, intracellular adaptations take place in the elastic properties of the giant muscle protein titin. We performed extensive single muscle fiber mechanical studies to determine active and passive properties in TNX-deficient EDS patients. Gel-electrophoresis, Western blotting, and microarray studies were used to evaluate titin expression and phosphorylation. X-ray diffraction was used tomore » measure myofilament lattice spacing. Passive tension of muscle fibers from TNX-deficient EDS patients was markedly increased. Myofilament extraction experiments indicated that the increased passive tension is attributable to changes in the properties of the sarcomeric protein titin. Transcript and protein data indicated no changes in titin isoform expression. Instead, differences in posttranslational modifications within titin's elastic region were found. In patients, active tension was not different at maximal activation level, but at submaximal activation level it was augmented attributable to increased calcium sensitivity. This increased calcium sensitivity might be attributable to stiffer titin molecules. In response to the increased compliance of the extracellular matrix in muscle of TNX-deficient EDS patients, a marked intracellular stiffening occurs of the giant protein titin. The stiffening of titin partly compensates for the muscle weakness in these patients by augmenting submaximal active tension generation.« less

High pH-Sensitive TRPA1 Activation in Odontoblasts Regulates Mineralization.

PubMed

Kimura, M; Sase, T; Higashikawa, A; Sato, M; Sato, T; Tazaki, M; Shibukawa, Y

2016-08-01

Calcium hydroxide and mineral trioxide aggregate are widely used for indirect and direct pulp capping and root canal filling. Their dissociation into Ca(2+) and OH(-) in dental pulp creates an alkaline environment, which activates reparative/reactionary dentinogenesis. However, the mechanisms by which odontoblasts detect the pH of the extracellular environment remain unclear. We examined the alkali-sensitive intracellular Ca(2+) signaling pathway in rat odontoblasts. In the presence or absence of extracellular Ca(2+), application of alkaline solution increased intracellular Ca(2+) concentration, or [Ca(2+)]i Alkaline solution-induced [Ca(2+)]i increases depended on extracellular pH (8.5 to 10.5) in both the absence and the presence of extracellular Ca(2+) The amplitude was smaller in the absence than in the presence of extracellular Ca(2+) Each increase in [Ca(2+)]i, activated by pH 7.5, 8.5, or 9.5, depended on extracellular Ca(2+) concentration; the equilibrium binding constant for extracellular Ca(2+) concentration decreased as extracellular pH increased (1.04 mM at pH 7.5 to 0.11 mM at pH 9.5). Repeated applications of alkaline solution did not have a desensitizing effect on alkali-induced [Ca(2+)]i increases and inward currents. In the presence of extracellular Ca(2+), alkaline solution-induced [Ca(2+)]i increases were suppressed by application of an antagonist of transient receptor potential ankyrin subfamily member 1 (TRPA1) channels. Ca(2+) exclusion efficiency during alkaline solution-induced [Ca(2+)]i increases was reduced by a Na(+)-Ca(2+) exchanger antagonist. Alizarin red and von Kossa staining revealed increased mineralization levels under repeated high pH stimulation, whereas the TRPA1 antagonist strongly reduced this effect. These findings indicate that alkaline stimuli-such as the alkaline environment inside dental pulp treated with calcium hydroxide or mineral trioxide aggregate-activate Ca(2+) mobilization via Ca(2+) influx mediated by TRPA1 channels and intracellular Ca(2+) release in odontoblasts. High pH-sensing mechanisms in odontoblasts are important for activating dentinogenesis induced by an alkaline environment. © International & American Associations for Dental Research 2016.

Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain

PubMed Central

Anderson, Joel G.; Fordahl, Steve C.; Cooney, Paula T.; Weaver, Tara L.; Colyer, Christa L.; Erikson, Keith M.

2011-01-01

Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently γ-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased 3H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABAA, and GABAB differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor proteins, which may be the basis of the neurological characteristics of manganism. PMID:18771689

Selective inhibition of dopamine-beta-hydroxylase enhances dopamine release from noradrenergic terminals in the medial prefrontal cortex.

PubMed

Devoto, Paola; Flore, Giovanna; Saba, Pierluigi; Frau, Roberto; Gessa, Gian L

2015-10-01

Disulfiram has been claimed to be useful in cocaine addiction therapy, its efficacy being attributed to dopamine-beta-hydroxylase (DBH) inhibition. Our previous results indicate that disulfiram and the selective DBH inhibitor nepicastat increase extracellular dopamine (DA) in the rat medial prefrontal cortex (mPFC), and markedly potentiated cocaine-induced increase. Concomitantly, in rats with cocaine self-administration history, cocaine-seeking behavior induced by drug priming was prevented, probably through overstimulation of D1 receptors due to the DA increase. The present research was aimed at studying the neurochemical mechanisms originating the enhanced DA release. Noradrenergic system ablation was attained by intracerebroventricular (i.c.v.) administration of the neurotoxin anti-DBH-saporin (aDBH-sap). DA, noradrenaline (NA), and DOPAC were assessed by HPLC after ex vivo tissue extraction or in vivo microdialysis. Control and denervated rats were subjected to microdialysis in the mPFC and caudate nucleus to evaluate the effect of nepicastat-cocaine combination on extracellular DA levels and their regulation by α2-adrenoceptors. Fifteen days after neurotoxin or its vehicle administration, tissue and extracellular NA were reduced to less than 2% the control value, while extracellular DA was increased by approximately 100%. In control rats, nepicastat given alone and in combination with cocaine increased extracellular DA by about 250% and 1100%, respectively. In denervated rats, nepicastat slightly affected extracellular DA, while in combination with cocaine increased extracellular DA by 250%. No differences were found in the caudate nucleus. Clonidine almost totally reversed the extracellular DA elevation produced by nepicastat-cocaine combination, while it was ineffective in denervated rats. This research shows that the increase of extracellular DA produced by nepicastat alone or in combination with cocaine was prevented by noradrenergic denervation. The results indicate that nepicastat enhances DA release from noradrenergic terminals supposedly by removing NA from α2-autoreceptors. In addition to the inhibition of DA uptake, the latter mechanism may explain the synergistic effect of cocaine on nepicastat-induced DA release.

Sexual odor preference and dopamine release in the nucleus accumbens by estrous olfactory cues in sexually naïve and experienced male rats.

PubMed

Fujiwara, Masaya; Chiba, Atsuhiko

2018-03-01

Sexual behavior is a natural reward that activates mesolimbic dopaminergic system. Microdialysis studies have shown that extracellular level of dopamine (DA) in the nucleus accumbens (NAcc) significantly increases during copulation in male rats. The NAcc DA level is also known to be increased during the presentation of a sexually receptive female before mating. This rise in DA was probably associated with sexual motivation elicited by incentive stimuli from the receptive female. These microdialysis studies, however, did not thoroughly investigated if olfactory stimuli from estrous females could significantly increase the extracellular DA in the NAcc of male rats. The present study was designed to examine systematically the relationship between the expression of preference for the olfactory stimuli from estrous females and the effects of these stimuli on the extracellular DA levels in the NAcc measured by in vivo microdialysis in male Long-Evans (LE) rats. We used two types of olfactory stimuli, either airborne odors (volatile stimuli) or soiled bedding (volatile plus nonvolatile stimuli). The sexually experienced male rats, which experienced six ejaculations, significantly preferred both of these olfactory stimuli from estrous females as opposed to males. Exposure to these female olfactory stimuli gradually increased extracellular DA in the NAcc, which reached significantly higher level above baseline during the period following the removal of the stimuli although not during the 15-min stimulus presentation period. The sexually naïve male rats, on the other hand, showed neither preference for olfactory stimuli from estrous females nor increase in the NAcc DA after exposure to these stimuli. These data suggest that in male LE rats olfactory stimuli from estrous females in and of themselves can be conditional cues that induce both incentive motivation and a significant increase in the NAcc DA probably as a result of being associated with sexual reward through copulatory experience. Copyright © 2017. Published by Elsevier Inc.

Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice.

PubMed

Kyriakides, T R; Zhu, Y H; Yang, Z; Huynh, G; Bornstein, P

2001-10-01

The matricellular angiogenesis inhibitor, thrombospondin (TSP) 2, has been shown to be an important modulator of wound healing and the foreign body response. Specifically, TSP2-null mice display improved healing with minimal scarring and form well-vascularized foreign body capsules. In this study we performed subcutaneous implantation of sponges and investigated the resulting angiogenic and fibrogenic responses. Histological and immunohistochemical analysis of sponges, excised at 7, 14, and 21 days after implantation, revealed significant differences between TSP2-null and wild-type mice. Most notably, TSP2-null mice exhibited increased angiogenesis and fibrotic encapsulation of the sponge. However, invasion of dense tissue was compromised, even though its overall density was increased. Furthermore, histomorphometry and biochemical assays demonstrated a significant increase in the extracellular distribution of matrix metalloproteinase (MMP) 2, but no change in the levels of active transforming growth factor-beta(1). The alterations in neovascularization, dense tissue invasion, and MMP2 in TSP2-null mice coincided with the deposition of TSP2 in the extracellular matrix of wild-type animals. These observations support the proposed role of TSP2 as a modulator of angiogenesis and matrix remodeling during tissue repair. In addition, they provide in vivo evidence for a newly proposed function of TSP2 as a modulator of extracellular MMP2 levels.

Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

PubMed Central

Liu, Jianming; Burkin, Dean J.; Kaufman, Stephen J.

2008-01-01

The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. PMID:18045857

Alterations in brain extracellular dopamine and glycine levels following combined administration of the glycine transporter type-1 inhibitor Org-24461 and risperidone.

PubMed

Nagy, Katalin; Marko, Bernadett; Zsilla, Gabriella; Matyus, Peter; Pallagi, Katalin; Szabo, Geza; Juranyi, Zsolt; Barkoczy, Jozsef; Levay, Gyorgy; Harsing, Laszlo G

2010-12-01

The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D(2) dopamine receptors. N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D(2) dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced dopaminergic side effects characteristic for antipsychotic medication.

Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

PubMed

Leo, Giuseppina; Guescini, Michele; Genedani, Susanna; Stocchi, Vilberto; Carone, Chiara; Filaferro, Monica; Sisti, Davide; Marcoli, Manuela; Maura, Guido; Cortelli, Pietro; Guidolin, Diego; Fuxe, Kjell; Agnati, Luigi Francesco

2015-04-01

Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state. Copyright © 2015 Elsevier Inc. All rights reserved.

The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane.

PubMed

Liu, Hengjun; Maruyama, Hisataka; Masuda, Taisuke; Honda, Ayae; Arai, Fumihito

2016-01-01

Influenza virus infection can result in changes in the cellular ion levels at 2-3 h post-infection. More H(+) is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H(+) during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H(+) from the intracellular compartment. Increased H(+) export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5-0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.

Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps.

PubMed

Jansen, Marcel P B; Emal, Diba; Teske, Gwendoline J D; Dessing, Mark C; Florquin, Sandrine; Roelofs, Joris J T H

2017-02-01

Acute kidney injury is often the result of ischemia reperfusion injury, which leads to activation of coagulation and inflammation, resulting in necrosis of renal tubular epithelial cells. Platelets play a central role in coagulation and inflammatory processes, and it has been shown that platelet activation exacerbates acute kidney injury. However, the mechanism of platelet activation during ischemia reperfusion injury and how platelet activation leads to tissue injury are largely unknown. Here we found that renal ischemia reperfusion injury in mice leads to increased platelet activation in immediate proximity of necrotic cell casts. Furthermore, platelet inhibition by clopidogrel decreased cell necrosis and inflammation, indicating a link between platelet activation and renal tissue damage. Necrotic tubular epithelial cells were found to release extracellular DNA, which, in turn, activated platelets, leading to platelet-granulocyte interaction and formation of neutrophil extracellular traps ex vivo. Renal ischemia reperfusion injury resulted in increased DNA-platelet and DNA-platelet-granulocyte colocalization in tissue and elevated levels of circulating extracellular DNA and platelet factor 4 in mice. After renal ischemia reperfusion injury, neutrophil extracellular traps were formed within renal tissue, which decreased when mice were treated with the platelet inhibitor clopidogrel. Thus, during renal ischemia reperfusion injury, necrotic cell-derived DNA leads to platelet activation, platelet-granulocyte interaction, and subsequent neutrophil extracellular trap formation, leading to renal inflammation and further increase in tissue injury. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzyme-based sensor.

PubMed

Hu, Y; Wilson, G S

1997-10-01

A successfully developed enzyme-based lactate microsensor with rapid response time allows the direct and continuous in vivo measurement of lactic acid concentration with high temporal resolution in brain extracellular fluid. The fluctuations coupled to neuronal activity in extracellular lactate concentration were explored in the dentate gyrus of the hippocampus of the rat brain after electrical stimulation of the perforant pathway. Extracellular glucose and oxygen levels were also detected simultaneously by coimplantation of a fast-response glucose sensor and an oxygen electrode, to provide novel information of trafficking of energy substances in real time related to local neuronal activity. The results first give a comprehensive picture of complementary energy supply and use of lactate and glucose in the intact brain tissue. In response to acute neuronal activation, the brain tissue shifts immediately to significant energy supply by lactate. A local temporary fuel "reservoir" is established behind the blood-brain barrier, evidenced by increased extracellular lactate concentration. The pool can be depleted rapidly, up to 28% in 10-12 s, by massive, acute neuronal use after stimulation and can be replenished in approximately 20 s. Glutamate-stimulated astrocytic glycolysis and the increase of regional blood flow may regulate the lactate concentration of the pool in different time scales to maintain local energy homeostasis.

Peripheral Administration of Ethanol Results in a Correlated Increase in Dopamine and Serotonin Within the Posterior Ventral Tegmental Area

PubMed Central

Deehan, Gerald A.; Knight, Christopher P.; Waeiss, R. Aaron; Engleman, Eric A.; Toalston, Jamie E.; McBride, William J.; Hauser, Sheketha R.; Rodd, Zachary A.

2016-01-01

Aims Two critical neurotransmitter systems regulating ethanol (EtOH) reward are serotonin (5-HT) and dopamine (DA). Within the posterior ventral tegmental area (pVTA), 5-HT receptors have been shown to regulate DA neuronal activity. Increased pVTA neuronal activity has been linked to drug reinforcement. The current experiment sought to determine the effect of EtOH on 5-HT and DA levels within the pVTA. Methods Wistar rats were implanted with cannula aimed at the pVTA. Neurochemical levels were determined using standard microdialysis procedures with concentric probes. Rats were randomly assigned to one of the five groups (n = 41; 7–9 per group) that were treated with 0–3.0 g/kg EtOH (intraperitoneally). Results Ethanol produced increased extracellular DA levels in the pVTA that resembled an inverted U-shape dose–response curve with peak levels (~200% of baseline) at the 2.25 g/kg dose. The increase in DA levels was observed for an extended period of time (~100 minutes). The effects of EtOH on extracellular 5-HT levels in the pVTA also resembled an inverted U-shape dose–response curve. However, increased 5-HT levels were only observed during the initial post-injection sample. The increases in extracellular DA and 5-HT levels were significantly correlated. Conclusion The data indicate intraperitoneal EtOH administration stimulated the release of both 5-HT and DA within the pVTA, the levels of which were significantly correlated. Overall, the current findings suggest that the ability of EtOH to stimulate DA activity within the mesolimbic system may be modulated by increases in 5-HT release within the pVTA. Short summary Two critical neurotransmitter systems regulating ethanol reward are serotonin and dopamine. The current experiment determined that intraperitoneal ethanol administration increased serotonin and dopamine levels within the pVTA (levels were significantly correlated). The current findings suggest the ability of EtOH to stimulate serotonin and dopamine activity within the mesolimbic system. PMID:27307055

Does abnormal glycogen structure contribute to increased susceptibility to seizures in epilepsy?

PubMed Central

DiNuzzo, Mauro; Mangia, Silvia; Maraviglia, Bruno; Giove, Federico

2014-01-01

Epilepsy is a family of brain disorders with a largely unknown etiology and high percentage of pharmacoresistance. The clinical manifestations of epilepsy are seizures, which originate from aberrant neuronal synchronization and hyperexcitability. Reactive astrocytosis, a hallmark of the epileptic tissue, develops into loss-of-function of glutamine synthetase, impairment of glutamate-glutamine cycle and increase in extracellular and astrocytic glutamate concentration. Here, we argue that chronically elevated intracellular glutamate level in astrocytes is instrumental to alterations in the metabolism of glycogen and leads to the synthesis of polyglucosans. Unaccessibility of glycogen-degrading enzymes to these insoluble molecules compromises the glycogenolysis-dependent reuptake of extracellular K+ by astrocytes, thereby leading to increased extracellular K+ and associated membrane depolarization. Based on current knowledge, we propose that the deterioration in structural homogeneity of glycogen particles is relevant to disruption of brain K+ homeostasis and increased susceptibility to seizures in epilepsy. PMID:24643875

Does abnormal glycogen structure contribute to increased susceptibility to seizures in epilepsy?

PubMed

DiNuzzo, Mauro; Mangia, Silvia; Maraviglia, Bruno; Giove, Federico

2015-02-01

Epilepsy is a family of brain disorders with a largely unknown etiology and high percentage of pharmacoresistance. The clinical manifestations of epilepsy are seizures, which originate from aberrant neuronal synchronization and hyperexcitability. Reactive astrocytosis, a hallmark of the epileptic tissue, develops into loss-of-function of glutamine synthetase, impairment of glutamate-glutamine cycle and increase in extracellular and astrocytic glutamate concentration. Here, we argue that chronically elevated intracellular glutamate level in astrocytes is instrumental to alterations in the metabolism of glycogen and leads to the synthesis of polyglucosans. Unaccessibility of glycogen-degrading enzymes to these insoluble molecules compromises the glycogenolysis-dependent reuptake of extracellular K(+) by astrocytes, thereby leading to increased extracellular K(+) and associated membrane depolarization. Based on current knowledge, we propose that the deterioration in structural homogeneity of glycogen particles is relevant to disruption of brain K(+) homeostasis and increased susceptibility to seizures in epilepsy.

In vivo substitution of choline for sodium evokes a selective osmoinsensitive increase of extracellular taurine in the rat hippocampus.

PubMed

Lehmann, A; Sandberg, M

1990-01-01

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.

Tyrosine - Effects on catecholamine release

NASA Technical Reports Server (NTRS)

Acworth, Ian N.; During, Matthew J.; Wurtman, Richard J.

1988-01-01

Tyrosine administration elevates striatal levels of dopamine metabolites in animals given treatments that accelerate nigrostriatal firing, but not in untreated rats. We examined the possibility that the amino acid might actually enhance dopamine release in untreated animals, but that the technique of measuring striatal dopamine metabolism was too insensitive to demonstrate such an effect. Dopamine release was assessed directly, using brain microdialysis of striatal extracellular fluid. Tyrosine administration (50-200 mg/kg IP) did indeed cause a dose related increase in extracellular fluid dopamine levels with minor elevations in levels of DOPAC and HVA, its major metabolites, which were not dose-related. The rise in dopamine was short-lived, suggesting that receptor-mediated feedback mechanisms responded to the increased dopamine release by diminishing neuronal firing or sensitivity to tyrosine. These observations indicate that measurement of changes in striatal DOPAC and HVA, if negative, need not rule out increases in nigrostriatal dopamine release.

Quantification of extracellular levels of corticosterone in the basolateral amygdaloid complex of freely-moving rats: a dialysis study of circadian variation and stress-induced modulation.

PubMed

Bouchez, Gaëlle; Millan, Mark J; Rivet, Jean-Michel; Billiras, Rodolphe; Boulanger, Raphaël; Gobert, Alain

2012-05-03

Corticosterone influences emotion and cognition via actions in a diversity of corticolimbic structures, including the amygdala. Since extracellular levels of corticosterone in brain have rarely been studied, we characterized a specific and sensitive enzymatic immunoassay for microdialysis quantification of corticosterone in the basolateral amygdaloid complex of freely-moving rats. Corticosterone levels showed marked diurnal variation with an evening (dark phase) peak and stable, low levels during the day (light phase). The "anxiogenic agents", FG7142 (20 mg/kg) and yohimbine (10 mg/kg), and an environmental stressor, 15-min forced-swim, induced marked and sustained (1-3 h) increases in dialysis levels of corticosterone in basolateral amygdaloid complex. They likewise increased dialysis levels of dopamine and noradrenaline, but not serotonin and GABA. As compared to basal corticosterone levels of ~200-300 pg/ml, the elevation provoked by forced-swim was ca. 20-fold and this increase was abolished by adrenalectomy. Interestingly, stress-induced rises of corticosterone levels in basolateral amygdaloid complex were abrogated by combined but not separate administration of the corticotrophin releasing factor(1) (CRF(1)) receptor antagonist, CP154,526, and the vasopressin(1b) (V(1b)) receptor antagonist, SSR149,415. Underpinning their specificity, they did not block forced-swim-induced elevations in dopamine and noradrenaline. In conclusion, extracellular levels of corticosterone in the basolateral amygdaloid complex display marked diurnal variation. Further, they are markedly elevated by acute stressors, the effects of which are mediated (in contrast to concomitant elevations in levels of monoamines) by co-joint recruitment of CRF(1) and V(1b) receptors. Copyright © 2012 Elsevier B.V. All rights reserved.

In vivo administration of extracellular cGMP normalizes TNF-α and membrane expression of AMPA receptors in hippocampus and spatial reference memory but not IL-1β, NMDA receptors in membrane and working memory in hyperammonemic rats.

PubMed

Cabrera-Pastor, Andrea; Hernandez-Rabaza, Vicente; Taoro-Gonzalez, Lucas; Balzano, Tiziano; Llansola, Marta; Felipo, Vicente

2016-10-01

Patients with hepatic encephalopathy (HE) show working memory and visuo-spatial orientation deficits. Hyperammonemia is a main contributor to cognitive impairment in HE. Hyperammonemic rats show impaired spatial learning and learning ability in the Y maze. Intracerebral administration of extracellular cGMP restores learning in the Y-maze. The underlying mechanisms remain unknown. It also remains unknown whether extracellular cGMP improves neuroinflammation or restores spatial learning in hyperammonemic rats and if it affects differently reference and working memory. The aims of this work were: Spatial working and reference memory were assessed using the radial and Morris water mazes and neuroinflammation by immunohistochemistry and Western blot. Membrane expression of NMDA and AMPA receptor subunits was analyzed using the BS3 crosslinker. Extracellular cGMP was administered intracerebrally using osmotic minipumps. Chronic hyperammonemia induces neuroinflammation in hippocampus, with astrocytes activation and increased IL-1β, which are associated with increased NMDA receptors membrane expression and impaired working memory. This process is not affected by extracellular cGMP. Hyperammonemia also activates microglia and increases TNF-α, alters membrane expression of AMPA receptor subunits (increased GluA1 and reduced GluA2) and impairs reference memory. All these changes are reversed by extracellular cGMP. These results show that extracellular cGMP modulates spatial reference memory but not working memory. This would be mediated by modulation of TNF-α levels and of membrane expression of GluA1 and GluA2 subunits of AMPA receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluctuations in Nucleus Accumbens Extracellular Glutamate and Glucose during Motivated Glucose-drinking Behavior: Dissecting the Neurochemistry of Reward

PubMed Central

Wakabayashi, Ken T.; Myal, Stephanie E.; Kiyatkin, Eugene A.

2015-01-01

While motivated behavior involves multiple neurochemical systems, few studies have focused on the role of glutamate, the brain’s excitatory neurotransmitter, and glucose, the energetic substrate of neural activity in reward-related neural processes. Here, we used high-speed amperometry with enzyme-based substrate-sensitive and control, enzyme-free biosensors to examine second-scale fluctuations in the extracellular levels of these substances in the nucleus accumbens shell during glucose-drinking behavior in trained rats. Glutamate rose rapidly after the presentation of a glucose-containing cup and before the initiation of drinking (reward seeking), decreased more slowly to levels below baseline during consumption (sensory reward), and returned to baseline when the ingested glucose reached the brain (metabolic reward). When water was substituted for glucose, glutamate rapidly increased with cup presentation and in contrast to glucose drinking, increased above baseline after rats tasted the water and refused to drink further. Therefore, extracellular glutamate show distinct changes associated with key events of motivated drinking behavior and opposite dynamics during sensory and metabolic components of reward. In contrast to glutamate, glucose increased at each stimulus and behavioral event, showing a sustained elevation during the entire behavior and a robust post-ingestion rise that correlated with the gradual return of glutamate levels to their baseline. By comparing active drinking with passive intra-gastric glucose delivery, we revealed that fluctuations in extracellular glucose are highly dynamic, reflecting a balance between rapid delivery due to neural activity, intense metabolism, and the influence of ingested glucose reaching the brain. PMID:25393775

The activation of metabotropic glutamate 5 receptors in the rat ventral tegmental area increases dopamine extracellular levels.

PubMed

Ferrada, Carla; Sotomayor-Zárate, Ramón; Abarca, Jorge; Gysling, Katia

2017-01-01

The mesocorticolimbic circuit projects to the prefrontal cortex, hippocampus, amygdala, and nucleus accumbens, among others, and it originates in the dopaminergic neurons of the ventral tegmental area (VTA). The VTA receives glutamatergic inputs from the prefrontal cortex and several subcortical regions. The glutamate released activates dopaminergic neurons and its action depends on the activation of ionotropic and metabotropic glutamate receptors. VTA dopaminergic neurons release dopamine (DA) from axon terminals in the innervated regions and somatodendritically in the VTA itself. DA release in the VTA is directly correlated with the activity of dopaminergic neurons. We hypothesized that metabotropic glutamate 5 receptors (mGlu5) directly regulate the activity of VTA dopaminergic neurons. To test this hypothesis, the extracellular levels of VTA DA and glutamate were studied by in-vivo microdialysis after an intra-VTA perfusion of (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG), selective mGlu5 agonist. We observed that CHPG induced a significant increase in VTA DA and glutamate extracellular levels. To determine whether the effect of CHPG on DA levels is because of the increase in glutamate release, we perfused kynurenic acid, an ionotropic glutamate receptor antagonist, through the probe. Our results showed that kynurenic acid did not block the ability of CHPG to cause DA release. Thus, our results suggest that CHPG acts directly on mGlu5 in dopaminergic neurons to induce the release of DA.

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue.

PubMed

Strouch, Marci B; Jackson, Edwin K; Mi, Zaichuan; Metes, Nicole A; Carey, Gale B

2005-06-01

Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

Comparative effects of parathion and chlorpyrifos on extracellular endocannabinoid levels in rat hippocampus: Influence on cholinergic toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Jing; Parsons, Loren; Pope, Carey, E-mail: carey.pope@okstate.edu

2013-11-01

Parathion (PS) and chlorpyrifos (CPF) are organophosphorus insecticides (OPs) that elicit acute toxicity by inhibiting acetylcholinesterase (AChE). Endocannabinoids (eCBs, N-arachidonoylethanolamine, AEA; 2-arachidonoylglycerol, 2AG) can modulate neurotransmission by inhibiting neurotransmitter release. We proposed that differential inhibition of eCB-degrading enzymes (fatty acid amide hydrolase, FAAH, and monoacylglycerol lipase, MAGL) by PS and CPF leads to differences in extracellular eCB levels and toxicity. Microdialysis cannulae were implanted into hippocampus of adult male rats followed by treatment with vehicle (peanut oil, 2 ml/kg, sc), PS (27 mg/kg) or CPF (280 mg/kg) 6–7 days later. Signs of toxicity, AChE, FAAH and MAGL inhibition, and extracellularmore » levels of AEA and 2AG were measured 2 and 4 days later. Signs were noted in PS-treated rats but not in controls or CPF-treated rats. Cholinesterase inhibition was extensive in hippocampus with PS (89–90%) and CPF (78–83%) exposure. FAAH activity was also markedly reduced (88–91%) by both OPs at both time-points. MAGL was inhibited by both OPs but to a lesser degree (35–50%). Increases in extracellular AEA levels were noted after either PS (about 2-fold) or CPF (about 3-fold) while lesser treatment-related 2-AG changes were noted. The cannabinoid CB1 receptor antagonist/inverse agonist AM251 (3 mg/kg, ip) had no influence on functional signs after CPF but markedly decreased toxicity in PS-treated rats. The results suggest that extracellular eCBs levels can be markedly elevated by both PS and CPF. CB1-mediated signaling appears to play a role in the acute toxicity of PS but the role of eCBs in CPF toxicity remains unclear. - Highlights: • Chlorpyrifos and parathion both extensively inhibited hippocampal cholinesterase. • Functional signs were only noted with parathion. • Chlorpyrifos and parathion increased hippocampal extracellular anandamide levels. • 2-Arachidonoylglycerol levels were lesser affected. • The CB1 antagonist AM251 had no effect on chlorpyrifos but reduced parathion toxicity.« less

Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex.

PubMed

Muschamp, John W; Regina, Meredith J; Hull, Elaine M; Winter, Jerrold C; Rabin, Richard A

2004-10-08

The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.

Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium.

PubMed

Morland, Cecilie; Pettersen, Mi Nguyen; Hassel, Bjørnar

2016-05-01

Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia. Copyright © 2016. Published by Elsevier B.V.

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

PubMed Central

Ferreira de Oliveira, José Miguel P.; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on d-sorbitol, small amounts of d-maltose or d-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by d-maltose or d-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on d-maltose and β-xylosidase D on d-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra d-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of d-xylose or d-maltose. Furthermore, d-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers d-maltose and d-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by d-maltose or d-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for d-xylose induction, d-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation. PMID:21698107

Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose.

PubMed

de Oliveira, José Miguel P Ferreira; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Mephedrone, compared with MDMA (ecstasy) and amphetamine, rapidly increases both dopamine and 5-HT levels in nucleus accumbens of awake rats

PubMed Central

Kehr, J; Ichinose, F; Yoshitake, S; Goiny, M; Sievertsson, T; Nyberg, F; Yoshitake, T

2011-01-01

BACKGROUND AND PURPOSE The designer drug 1-(4-methylphenyl)-2-methylaminopropan-1-one (4-methylmethcathinone, mephedrone) is reported to possess psychostimulant, entactogenic and hallucinogenic effects. The purpose of this study was to examine the effects of acute administration of mephedrone on extracellular levels of dopamine (DA) and 5-HT in the nucleus accumbens of awake rats and compare these effects with those induced by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and amphetamine. EXPERIMENTAL APPROACH Microdialysis sampling was performed while simultaneously recording locomotor activity in rats and the monoamines were determined by HPLC with electrochemical detection. KEY RESULTS Mephedrone (3 mg·kg−1 s.c.) and (+)-amphetamine (1 mg·kg−1 s.c.) caused rapid increases in extracellular DA levels of 496% and 412%, respectively, whereas MDMA (3 mg·kg−1 s.c.) showed only a moderate effect (235%). The corresponding 5-HT levels increased to 941% (mephedrone) and 911% (MDMA), but only to 165% following amphetamine. The calculated t1/2 values for elimination rate of mephedrone, MDMA and amphetamine-induced increases in extracellular DA levels were 25, 303 and 51 min, the corresponding t1/2 values for 5-HT were 26, 48 and 84 min, respectively. Locomotor activity was increased most by amphetamine, whereas both mephedrone and MDMA showed about three times lower and shorter-lasting effects. CONCLUSIONS AND IMPLICATIONS The neurochemical and functional properties of mephedrone resemble those of MDMA, but it also shows an amphetamine-like effect in that it evokes a rapid release and elimination of DA in the brain reward system, a feature that may contribute to its potent re-inforcing properties. PMID:21615721

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

PubMed

Park, Gunhyuk; Oh, Dal-Seok; Kim, Yong-Ung; Park, Moon Ki

2017-01-01

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70. © 2017 S. Karger AG, Basel.

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

PubMed

Sturm, A; Chrispeels, M J

1990-11-01

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

PubMed Central

Sturm, A; Chrispeels, M J

1990-01-01

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose. PMID:2152110

Effects of local infusions of apomorphine on the extracellular citrulline level in the striatum: Involvement of D1 and D2 dopamine receptors.

PubMed

Savel'ev, S A

2006-11-01

Studies using vital microdialysis and high-performance liquid chromatography showed that local infusion of the NO synthase inhibitor N-nitro-L-arginine (1 mM) into the striatum decreased, while infusion of the dopamine receptor agonist apomorphine (100 microM) increased the level of citrulline (a side product of nitric oxide synthesis) in the intercellular space of this structure in Sprague-Dawley rats. The increase in the citrulline level induced by infusions of apomorphine was completely prevented by local infusions of N-nitro-L-arginine (1 mM) and raclopride (10 microm), a dopamine D2 receptor blocker, but not by infusion of SCH-23390 (50 microm), a dopamine D1 receptor blocker. These data suggest that the increase in extracellular citrulline in the striatum induced by dopaminergic stimulation results from local increases in NO synthase activity and that this effect involves D2, but not D1 dopamine receptors.

High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation.

PubMed

Kitada, Kento; Daub, Steffen; Zhang, Yahua; Klein, Janet D; Nakano, Daisuke; Pedchenko, Tetyana; Lantier, Louise; LaRocque, Lauren M; Marton, Adriana; Neubert, Patrick; Schröder, Agnes; Rakova, Natalia; Jantsch, Jonathan; Dikalova, Anna E; Dikalov, Sergey I; Harrison, David G; Müller, Dominik N; Nishiyama, Akira; Rauh, Manfred; Harris, Raymond C; Luft, Friedrich C; Wassermann, David H; Sands, Jeff M; Titze, Jens

2017-05-01

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter-driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation

PubMed Central

Kitada, Kento; Daub, Steffen; Zhang, Yahua; Klein, Janet D.; Nakano, Daisuke; Pedchenko, Tetyana; Lantier, Louise; LaRocque, Lauren M.; Marton, Adriana; Neubert, Patrick; Schröder, Agnes; Rakova, Natalia; Jantsch, Jonathan; Dikalova, Anna E.; Dikalov, Sergey I.; Harrison, David G.; Müller, Dominik N.; Nishiyama, Akira; Rauh, Manfred; Harris, Raymond C.; Luft, Friedrich C.; Wasserman, David H.; Sands, Jeff M.

2017-01-01

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter–driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions. PMID:28414295

Contributions of the Na+/K+-ATPase, NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

PubMed Central

Larsen, Brian Roland; Assentoft, Mette; Cotrina, Maria L.; Hua, Susan Z.; Nedergaard, Maiken; Kaila, Kai; Voipio, Juha; MacAulay, Nanna

2015-01-01

Bursts of network activity in the brain are associated with a transient increase in extracellular K+ concentration. The excess K+ is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na+/K+/2Cl− cotransporter (NKCC1), and/or Na+/K+-ATPase activity. Their individual contribution to [K+]o management has been of extended controversy. The present study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na+/K+-ATPase and to resolve their involvement in clearance of extracellular K+ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K+]o increases above basal levels. Increased [K+]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K+ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K+ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K+]o increase. In contrast, inhibition of the different isoforms of Na+/K+-ATPase reduced post-stimulusclearance of K+ transients. The glia-specific α2/β2 subunit composition of Na+/K+-ATPase, when expressed in Xenopus oocytes, displayed a K+ affinity and voltage-sensitivity that would render this astrocyte-specific subunit composition specifically geared for controlling [K+]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na+/K+-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K+ recovery in native hippocampal tissue while Kir4.1 and Na+/K+-ATPase serve temporally distinct roles. PMID:24482245

[Human body composition during extended stay in microgravity].

PubMed

Noskov, V B; Nichiporuk, I A; Vasilieva, G Yu; Smirnov, Yu I

2015-01-01

According to the Sprut-2 protocol, bio-impedancemetry of ISS cosmonauts was performed once a month and also before and after mission. Multiple non-invasive body measurements were carried out in 15 cosmonauts in real time. Relocation of extracellular liquid along the body axis led to its reduction in legs and, on the contrary, an increase in the abdomen. Volumes of total body liquid as well as intra- and extracellular liquids decreased in comparison with pre-flight levels. Lean body mass also became less in microgravity, whereas fat mass showed an increase.

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

NASA Technical Reports Server (NTRS)

Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

2003-01-01

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Implications for glycine receptors and astrocytes in ethanol-induced elevation of dopamine levels in the nucleus accumbens.

PubMed

Adermark, Louise; Clarke, Rhona B C; Olsson, Torsten; Hansson, Elisabeth; Söderpalm, Bo; Ericson, Mia

2011-01-01

Elevated dopamine levels are believed to contribute to the rewarding sensation of ethanol (EtOH), and previous research has shown that strychnine-sensitive glycine receptors in the nucleus accumbens (nAc) are involved in regulating dopamine release and in mediating the reinforcing effects of EtOH. Furthermore, the osmoregulator taurine, which is released from astrocytes treated with EtOH, can act as an endogenous ligand for the glycine receptor, and increase extracellular dopamine levels. The aim of this study was to address if EtOH-induced swelling of astrocytes could contribute to elevated dopamine levels by increasing the extracellular concentration of taurine. Cell swelling was estimated by optical sectioning of fluorescently labeled astrocytes in primary cultures from rat, and showed that EtOH (25-150 mM) increased astrocyte cell volumes in a concentration- and ion-dependent manner. The EtOH-induced cell swelling was inhibited in cultures treated with the Na(+) /K(+) /2Cl⁻ cotransporter blocker furosemide (1 mM), Na(+) /K(+) -ATPase inhibitor ouabain (0.1 mM), potassium channel inhibitor BaCl₂ (50 µM) and in cultures containing low extracellular sodium concentration (3 mM). In vivo microdialysis performed in the nAc of awake and freely moving rats showed that local treatment with EtOH enhanced the concentrations of dopamine and taurine in the microdialysate, while glycine and β-alanine levels were not significantly modulated. EtOH-induced dopamine release was antagonized by local treatment with the glycine receptor antagonist strychnine (20 µM) or furosemide (100 µM or 1 mM). Furosemide also prevented EtOH-induced taurine release in the nAc. In conclusion, our data suggest that extracellular concentrations of dopamine and taurine are interconnected and that swelling of astrocytes contributes to the acute rewarding sensation of EtOH. © 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction.

Enhancement of cortical extracellular 5-HT by 5-HT1A and 5-HT2C receptor blockade restores the antidepressant-like effect of citalopram in non-responder mice.

PubMed

Calcagno, Eleonora; Guzzetti, Sara; Canetta, Alessandro; Fracasso, Claudia; Caccia, Silvio; Cervo, Luigi; Invernizzi, Roberto W

2009-07-01

We recently found that the response of DBA/2 mice to SSRIs in the forced swim test (FST) was impaired and they also had a smaller basal and citalopram-stimulated increase in brain extracellular serotonin (5-HT) than 'responder' strains. We employed intracerebral microdialysis, FST and selective antagonists of 5-HT1A and 5-HT2C receptors to investigate whether enhancing the increase in extracellular 5-HT reinstated the anti-immobility effect of citalopram in the FST. WAY 100635 (0.3 mg/kg s.c.) or SB 242084 (1 mg/kg s.c.), respectively a selective 5-HT1A and 5-HT2C receptor antagonist, raised the effect of citalopram (5 mg/kg) on extracellular 5-HT in the medial prefrontal cortex of DBA/2N mice (citalopram alone 5.2+/-0.3 fmol/20 microl, WAY 100635+citalopram 9.9+/-2.1 fmol/20 microl, SB 242084+ citalopram 7.6+/-1.0 fmol/20 microl) to the level reached in 'responder' mice given citalopram alone. The 5-HT receptor antagonists had no effect on the citalopram-induced increase in extracellular 5-HT in the dorsal hippocampus. The combination of citalopram with WAY 100635 or SB 242084 significantly reduced immobility time in DBA/2N mice that otherwise did not respond to either drug singly. Brain levels of citalopram in mice given citalopram alone or with 5-HT antagonists did not significantly differ. The results confirm that impaired 5-HT transmission accounts for the lack of effect of citalopram in the FST and suggest that enhancing the effect of SSRIs on extracellular 5-HT, through selective blockade of 5-HT1A and 5-HT2C receptors, could be a useful strategy to restore the response in treatment-resistant depression.

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

PubMed Central

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L.; Evans, Charles R.; Brenner, Charles

2011-01-01

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins. PMID:21589930

Participation of hippocampal agmatine in spatial learning: an in vivo microdialysis study.

PubMed

Rushaidhi, Madihah; Jing, Yu; Zhang, Hu; Liu, Ping

2013-02-01

Agmatine, decarboxylated arginine, is widely distributed in mammalian brains and is considered as a novel putative neurotransmitter. Recent research demonstrates spatial learning-induced increases in agmatine in memory-related structures at the tissue and presynaptic terminal levels. By using the in vivo microdialysis technique coupled with highly sensitive liquid chromatography/mass spectrometry assay, we investigated dynamic changes of extracellular agmatine in the rat dorsal hippocampus before, during and after water maze training to find a fixed hidden platform on the first and forth day of testing. It was firstly noted that the basal level of extracellular agmatine was significantly elevated on day 4. While swimming per se had no effect, a rapid rise (2-6 folds) in extracellular agmatine was observed during water maze training regardless of testing day. Such learning-induced rise was found to successively lessen across the multiple blocks of training on day 1. However, this pattern was reversed on day 4 when the platform was removed during the final training trial. The present study, for the first time, demonstrates water maze training-induced increase of extracellular agmatine in the dorsal hippocampus. The results suggest a role of endogenous agmatine in the encoding and retrieval of spatial information. Copyright © 2012 Elsevier Ltd. All rights reserved.

Microdialysate concentration changes do not provide sufficient information to evaluate metabolic effects of lactate supplementation in brain-injured patients

PubMed Central

Rothman, Douglas L; Nordström, Carl-Henrik

2016-01-01

Cerebral microdialysis is a widely used clinical tool for monitoring extracellular concentrations of selected metabolites after brain injury and to guide neurocritical care. Extracellular glucose levels and lactate/pyruvate ratios have high diagnostic value because they can detect hypoglycemia and deficits in oxidative metabolism, respectively. In addition, patterns of metabolite concentrations can distinguish between ischemia and mitochondrial dysfunction, and are helpful to choose and evaluate therapy. Increased intracranial pressure can be life-threatening after brain injury, and hypertonic solutions are commonly used for pressure reduction. Recent reports have advocated use of hypertonic sodium lactate, based on claims that it is glucose sparing and provides an oxidative fuel for injured brain. However, changes in extracellular concentrations in microdialysate are not evidence that a rise in extracellular glucose level is beneficial or that lactate is metabolized and improves neuroenergetics. The increase in glucose concentration may reflect inhibition of glycolysis, glycogenolysis, and pentose phosphate shunt pathway fluxes by lactate flooding in patients with mitochondrial dysfunction. In such cases, lactate will not be metabolizable and lactate flooding may be harmful. More rigorous approaches are required to evaluate metabolic and physiological effects of administration of hypertonic sodium lactate to brain-injured patients. PMID:27604313

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

PubMed

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

2011-05-11

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

Extracellular Alkalinization as a Defense Response in Potato Cells.

PubMed

Moroz, Natalia; Fritch, Karen R; Marcec, Matthew J; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

2017-01-01

A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists ( Phytophthora infestans and Spongospora subterranea ) and fungi ( Verticillium dahliae and Colletotrichum coccodes ). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

PubMed

Uchida, Sae; Kagitani, Fusako

2017-05-12

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

Extracellular chelation of zinc does not affect hippocampal excitability and seizure-induced cell death in rats

PubMed Central

Lavoie, Nathalie; Peralta, Modesto R; Chiasson, Marilou; Lafortune, Kathleen; Pellegrini, Luca; Seress, László; Tóth, Katalin

2007-01-01

In the nervous system, zinc can influence synaptic responses and at extreme concentrations contributes to epileptic and ischaemic neuronal injury. Zinc can originate from synaptic vesicles, the extracellular space and from intracellular stores. In this study, we aimed to determine which of these zinc pools is responsible for the increased hippocampal excitability observed in zinc-depleted animals or following zinc chelation. Also, we investigated the source of intracellularly accumulating zinc in vulnerable neurons. Our data show that membrane-permeable and membrane-impermeable zinc chelators had little or no effect on seizure activity in the CA3 region. Furthermore, extracellular zinc chelation could not prevent the accumulation of lethal concentrations of zinc in dying neurons following epileptic seizures. At the electron microscopic level, zinc staining significantly increased at the presynaptic membrane of mossy fibre terminals in kainic acid-treated animals. These data indicate that intracellular but not extracellular zinc chelators could influence neuronal excitability and seizure-induced zinc accumulation observed in the cytosol of vulnerable neurons. PMID:17095563

Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca(2+) signals and an IL-6 autocrine loop.

PubMed

Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique; Buvinic, Sonja

2014-04-15

Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

Fluctuations in nucleus accumbens extracellular glutamate and glucose during motivated glucose-drinking behavior: dissecting the neurochemistry of reward.

PubMed

Wakabayashi, Ken T; Myal, Stephanie E; Kiyatkin, Eugene A

2015-02-01

While motivated behavior involves multiple neurochemical systems, few studies have focused on the role of glutamate, the brain's excitatory neurotransmitter, and glucose, the energetic substrate of neural activity in reward-related neural processes. Here, we used high-speed amperometry with enzyme-based substrate-sensitive and control, enzyme-free biosensors to examine second-scale fluctuations in the extracellular levels of these substances in the nucleus accumbens shell during glucose-drinking behavior in trained rats. Glutamate rose rapidly after the presentation of a glucose-containing cup and before the initiation of drinking (reward seeking), decreased more slowly to levels below baseline during consumption (sensory reward), and returned to baseline when the ingested glucose reached the brain (metabolic reward). When water was substituted for glucose, glutamate rapidly increased with cup presentation and in contrast to glucose drinking, increased above baseline after rats tasted the water and refused to drink further. Therefore, extracellular glutamate show distinct changes associated with key events of motivated drinking behavior and opposite dynamics during sensory and metabolic components of reward. In contrast to glutamate, glucose increased at each stimulus and behavioral event, showing a sustained elevation during the entire behavior and a robust post-ingestion rise that correlated with the gradual return of glutamate levels to their baseline. By comparing active drinking with passive intra-gastric glucose delivery, we revealed that fluctuations in extracellular glucose are highly dynamic, reflecting a balance between rapid delivery because of neural activity, intense metabolism, and the influence of ingested glucose reaching the brain. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamura, Hitomi; Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501; Nio, Yasunori, E-mail: yasunori.nio@takeda.com

Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturatedmore » fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.« less

The involvement of brain-derived neurotrophic factor in 3,4-methylenedioxymethamphetamine-induced place preference and behavioral sensitization.

PubMed

Mouri, Akihiro; Noda, Yukihiro; Niwa, Minae; Matsumoto, Yurie; Mamiya, Takayoshi; Nitta, Atsumi; Yamada, Kiyofumi; Furukawa, Shoei; Iwamura, Tatsunori; Nabeshima, Toshitaka

2017-06-30

3,4-Methylenedioxymethamphetamine (MDMA) is known to induce dependence and psychosis in humans. Brain-derived neurotrophic factor (BDNF) is involved in the synaptic plasticity and neurotrophy in midbrain dopaminergic neurons. This study aimed to investigate the role of BDNF in MDMA-induced dependence and psychosis. A single dose of MDMA (10mg/kg) induced BDNF mRNA expression in the prefrontal cortex, nucleus accumbens, and amygdala, but not in the striatum or the hippocampus. However, repeated MDMA administration for 7 days induced BDNF mRNA expression in the striatum and hippocampus. Both precursor and mature BDNF protein expression increased in the nucleus accumbens, mainly in the neurons. Additionally, rapidly increased extracellular serotonin levels and gradually and modestly increased extracellular dopamine levels were noted within the nucleus accumbens of mice after repeated MDMA administration. Dopamine receptor antagonists attenuated the effect of repeated MDMA administration on BDNF mRNA expression in the nucleus accumbens. To examine the role of endogenous BDNF in the behavioral and neurochemical effects of MDMA, we used mice with heterozygous deletions of the BDNF gene. MDMA-induced place preference, behavioral sensitization, and an increase in the levels of extracellular serotonin and dopamine within the nucleus accumbens, were attenuated in BDNF heterozygous knockout mice. These results suggest that BDNF is implicated in MDMA-induced dependence and psychosis by activating the midbrain serotonergic and dopaminergic neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

Chromatographic analysis of age-related changes in mucosal serotonin transmission in the murine distal ileum

PubMed Central

2012-01-01

Background In the upper bowel, alterations in motility and absorption of key nutrients have been observed as part of the normal ageing process. Serotonin (5-HT) is a key signalling molecule in the gastrointestinal tract and is known to influence motility, however little is known of how the ageing process alters 5-HT signalling processes in the bowel. Results An isocratic chromatographic method was able to detect all 5-HT precursors and metabolites. Using extracellular and intracellular sampling approaches, we were able to monitor all key parameters associated with the transmission process. There was no alteration in the levels of tryptophan and 5-HTP between 3 and 18 month old animals. There was a significant increase in the ratio of 5-HT:5-HTP and an increase in intracellular 5-HT between 3 and 18 month old animals suggesting an increase in 5-HT synthesis. There was also a significant increase in extracellular 5-HT with age, suggesting increased 5-HT release. There was an age-related decrease in the ratio of intracellular 5-HIAA:extracellular 5-HT, whilst the amount of 5-HIAA did not change with age. In the presence of an increase in extracellular 5-HT, the lack of an age-related change in 5-HIAA is suggestive of a decrease in re-uptake via the serotonin transporter (SERT). Conclusions We have used intracellular and extracellular sampling to provide more insight into alterations in the neurotransmission process of 5-HT during normal ageing. We observed elevated 5-HT synthesis and release and a possible decrease in the activity of SERT. Taken together these changes lead to increased 5-HT availability and may alter motility function and could lead to the changes in adsorption observed in the elderly. PMID:22494644

On the Origin of Cortical Dopamine: Is it a Co-Transmitter in Noradrenergic Neurons?

PubMed Central

Devoto, Paola; Flore, Giovanna

2006-01-01

Dopamine (DA) and noradrenaline (NA) in the prefrontal cortex (PFC) modulate superior cognitive functions, and are involved in the aetiology of depressive and psychotic symptoms. Moreover, microdialysis studies in rats have shown how pharmacological treatments that induce modifications of extracellular NA in the medial PFC (mPFC), also produce parallel changes in extracellular DA. To explain the coupling of NA and DA changes, this article reviews the evidence supporting the hypothesis that extracellular DA in the cerebral cortex originates not only from dopaminergic terminals but also from noradrenergic ones, where it acts both as precursor for NA and as a co-transmitter. Accordingly, extracellular DA concentration in the occipital, parietal and cerebellar cortex was found to be much higher than expected in view of the scarce dopaminergic innervation in these areas. Systemic administration or intra-cortical perfusion of α2-adrenoceptor agonists and antagonists, consistent with their action on noradrenergic neuronal activity, produced concomitant changes not only in extracellular NA but also in DA in the mPFC, occipital and parietal cortex. Chemical modulation of the locus coeruleus by locally applied carbachol, kainate, NMDA or clonidine modified both NA and DA in the mPFC. Electrical stimulation of the locus coeruleus led to an increased efflux of both NA and DA in mPFC, parietal and occipital cortex, while in the striatum, NA efflux alone was enhanced. Atypical antipsychotics, such as clozapine and olanzapine, or antidepressants, including mirtazapine and mianserine, have been found to increase both NA and DA throughout the cerebral cortex, likely through blockade of α2-adrenoceptors. On the other hand, drugs selectively acting on dopaminergic transmission produced modest changes in extracellular DA in mPFC, and had no effect on the occipital or parietal cortex. Acute administration of morphine did not increase DA levels in the PFC (where NA is diminished), in contrast with augmented dopaminergic neuronal activity; moreover, during morphine withdrawal both DA and NA levels increased, in spite of a diminished dopaminergic activity, both increases being antagonised by clonidine but not quinpirole administration. Extensive 6-hydroxy dopamine lesion of the ventral tegmental area (VTA) decreases below 95% of control both intra- and extracellular DA and DOPAC in the nucleus accumbens, but only partially or not significantly in the mPFC and parietal cortex. The above evidence points to a common origin for NA and DA in the cerebral cortex and suggests the possible utility of noradrenergic system modulation as a target for drugs with potential clinical efficacy on cognitive functions. PMID:18615131

On the origin of cortical dopamine: is it a co-transmitter in noradrenergic neurons?

PubMed

Devoto, Paola; Flore, Giovanna

2006-04-01

Dopamine (DA) and noradrenaline (NA) in the prefrontal cortex (PFC) modulate superior cognitive functions, and are involved in the aetiology of depressive and psychotic symptoms. Moreover, microdialysis studies in rats have shown how pharmacological treatments that induce modifications of extracellular NA in the medial PFC (mPFC), also produce parallel changes in extracellular DA.To explain the coupling of NA and DA changes, this article reviews the evidence supporting the hypothesis that extracellular DA in the cerebral cortex originates not only from dopaminergic terminals but also from noradrenergic ones, where it acts both as precursor for NA and as a co-transmitter.Accordingly, extracellular DA concentration in the occipital, parietal and cerebellar cortex was found to be much higher than expected in view of the scarce dopaminergic innervation in these areas.Systemic administration or intra-cortical perfusion of alpha(2)-adrenoceptor agonists and antagonists, consistent with their action on noradrenergic neuronal activity, produced concomitant changes not only in extracellular NA but also in DA in the mPFC, occipital and parietal cortex.Chemical modulation of the locus coeruleus by locally applied carbachol, kainate, NMDA or clonidine modified both NA and DA in the mPFC.Electrical stimulation of the locus coeruleus led to an increased efflux of both NA and DA in mPFC, parietal and occipital cortex, while in the striatum, NA efflux alone was enhanced.Atypical antipsychotics, such as clozapine and olanzapine, or antidepressants, including mirtazapine and mianserine, have been found to increase both NA and DA throughout the cerebral cortex, likely through blockade of alpha(2)-adrenoceptors. On the other hand, drugs selectively acting on dopaminergic transmission produced modest changes in extracellular DA in mPFC, and had no effect on the occipital or parietal cortex.Acute administration of morphine did not increase DA levels in the PFC (where NA is diminished), in contrast with augmented dopaminergic neuronal activity; moreover, during morphine withdrawal both DA and NA levels increased, in spite of a diminished dopaminergic activity, both increases being antagonised by clonidine but not quinpirole administration.Extensive 6-hydroxy dopamine lesion of the ventral tegmental area (VTA) decreases below 95% of control both intra- and extracellular DA and DOPAC in the nucleus accumbens, but only partially or not significantly in the mPFC and parietal cortex.The above evidence points to a common origin for NA and DA in the cerebral cortex and suggests the possible utility of noradrenergic system modulation as a target for drugs with potential clinical efficacy on cognitive functions.

The changes of proteins and polysaccharides in extracellular polymeric substance for Spirogyra fluviatilis under different salinity

NASA Astrophysics Data System (ADS)

Lee, Yichao; Chang, Shuiping

2017-05-01

Spirogyra is a genus of widely distributed, large green fresh water algae. This study discovered that changes in salinity can induce Spirogyra fluviatilis to produce amounts of extracellular polymeric substance (EPS) when controlling other environmental conditions. If culturing S. fluviatilis with salinity greater than a 3.0‰ medium for 4 hours, the secretion EPS will be changed. And the level of polysaccharides and proteins, the primary components of EPS, is slightly increased in accordance with the increase in the salinity. But the proteins to polysaccharides ratio changes are not significantly

Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells.

PubMed

Bertoni, Ana Paula Santin; de Campos, Rafael Paschoal; Tsao, Marisa; Braganhol, Elizandra; Furlanetto, Tania Weber; Wink, Márcia Rosângela

2018-02-17

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

Nontypeable Haemophilus influenzae Induces Sustained Lung Oxidative Stress and Protease Expression

PubMed Central

King, Paul T.; Sharma, Roleen; O’Sullivan, Kim; Selemidis, Stavros; Lim, Steven; Radhakrishna, Naghmeh; Lo, Camden; Prasad, Jyotika; Callaghan, Judy; McLaughlin, Peter; Farmer, Michael; Steinfort, Daniel; Jennings, Barton; Ngui, James; Broughton, Bradley R. S.; Thomas, Belinda; Essilfie, Ama-Tawiah; Hickey, Michael; Holmes, Peter W.; Hansbro, Philip; Bardin, Philip G.; Holdsworth, Stephen R.

2015-01-01

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps. PMID:25793977

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain

PubMed Central

van der Vos, Kristan E.; Abels, Erik R.; Zhang, Xuan; Lai, Charles; Carrizosa, Esteban; Oakley, Derek; Prabhakar, Shilpa; Mardini, Osama; Crommentuijn, Matheus H. W.; Skog, Johan; Krichevsky, Anna M.; Stemmer-Rachamimov, Anat; Mempel, Thorsten R.; El Khoury, Joseph; Hickman, Suzanne E.; Breakefield, Xandra O.

2016-01-01

Background To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. Methods Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. Results Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. Conclusions Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs. PMID:26433199

Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-β by affecting insulin-degrading enzyme.

PubMed

Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Haupenthal, Viola J; Blümel, Tamara; Stötzel, Hannah; Grimm, Heike S; Hartmann, Tobias

2016-12-01

Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-β (Aβ) peptides as extracellular senile plaques in the brain. Total Aβ levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aβ-clearance mechanisms, including Aβ-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aβ-degradation by affecting insulin-degrading enzyme (IDE), the major Aβ-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aβ-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aβ production, EPA and DHA might ameliorate AD pathology by increasing Aβ turnover.

Near infrared laser irradiation induces NETosis via oxidative stress and autophagy.

PubMed

Mario, Migliario; Stelvio, Tonello; Vincenzo, Rochetti; Manuela, Rizzi; Filippo, Renò

2018-06-02

NETosis is a novel immune defense strategy in which neutrophil activation results in the formation of extracellular DNA/protein network which is able to kill microbial populations. NETosis can be induced in vitro by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA). Due to the importance of NETosis in different physiological and pathological processes, photobiostimulation effect on this neutrophil activation mechanism has been investigated. Human granulocytes, isolated from venous blood of healthy donors, were stimulated with a diode laser emitting at 980 nm with an energy intensity ranging from 0 to 75 joules. After 3 h of laser stimulation, granulocytes were fixed and colored with crystal violet in order to assess the NETosis morphology while extracellular DNA produced has been quantified using Sytox Green fluorescent dye. To evaluate ROS production and autophagy role in photobiostimulation-induced NETosis, granulocytes were pre-treated with ROS scavengers (vitamin C, sodium pyruvate, L-NAME, sodium azide), and an autophagy inhibitor (wortmannin). Laser stimulation induced an energy-dependent neutrophil extracellular trap (NET) production in human granulocytes starting from 50-J laser intensity. ROS scavengers and the autophagy inhibitor were able to abrogate both morphological features of NETosis and extracellular DNA production without modifying the basal level of NETosis. Photobiostimulation induced an increase in NET production due to an increase in ROS levels and autophagy activation.

[Change in concentration of cytosolic Ca2+ caused by extracellular ATP and ecto-ATP-ase activity in thymocytes and transformed MT-4 cells].

PubMed

Hrebinyk, S M; Artemenko, O Iu; Hryniuk, I I; Perepelitsyna, O M; Matyshevs'ka, O P

2009-01-01

The comparative study of extracellular ATP (ATP0) effect on free cytosolic calcium concentration ([Ca2+]i) in normal (isolated rat thymocytes) and transformed (leukosis MT-4 line) T-cells was carried out. Addition of 1 mM ATP to Ca-free incubation medium of both types of cells, loaded with indo-1, had no effect on [Ca2+]i level. Upon subsequent addition of 1 mM CaCl2 to the incubation medium the rapid and significant increase of [Ca2+]i in MT-4 cells was registered. This effect was maintained within 10 min and was not inhibited by phospholipase C inhibitor 0.2 mM neomycin, that was induced by cation entry into the cells from the extracellular medium. Both types of cells were shown to demonstrate ecto-ATPase activity in the presence of 1 mM MgCl2 or CaC12 in the incubation medium. Estimation of kinetic parameters has indicated that the maximum rate of extracellular ATP hydrolysis by MT-4 cells is higher and Mg2+ and Ca2+ activation constants are lower as compared to respective parameters of ATP hydrolysis by thymocytes. The possible functional significance of the increased level of ecto-ATPase activity in malignantly transformed cells is discussed.

Amphetamine regulation of acetylcholine and gamma-aminobutyric acid in nucleus accumbens.

PubMed

Lindefors, N; Hurd, Y L; O'Connor, W T; Brené, S; Persson, H; Ungerstedt, U

1992-01-01

In situ hybridization histochemistry and in vivo microdialysis were combined to study the effect of amphetamine on the expression of choline acetyltransferase and glutamate decarboxylase67 mRNA and in vivo release of acetylcholine and GABA in rat medial nucleus accumbens. Differential effects on acetylcholine and GABA neurons by a single challenge injection of amphetamine (1.5 mg/kg, s.c.) were apparent in saline-pretreated and amphetamine-pretreated (same dose, twice daily for the previous seven days) rats. Extracellular acetylcholine levels were increased up to 50% over a prolonged period following both single and repeated amphetamine. In contrast, extracellular concentrations of GABA were gradually decreased to half the control values, but only in rats receiving repeated amphetamine. Although the increase of acetylcholine release was not associated with any change in choline acetyltransferase mRNA levels, the number of neurons expressing high levels of glutamate decarboxylase67 mRNA was decreased (28%) following repeated injections. Thus we suggest that amphetamine decreases extracellular GABA levels by a slow mechanism, associated with the decreased expression of glutamate decarboxylase67 mRNA in a subpopulation of densely labeled neurons in the medial nucleus accumbens. The delayed response by GABA to amphetamine may reflect supersensitivity in the activity of postsynaptic gamma-aminobutyric acid-containing neurons in nucleus accumbens as a consequence of the repeated amphetamine treatment.

Extracellular H+ fluxes from tiger salamander Müller (glial) cells measured using self-referencing H+-selective microelectrodes.

PubMed

Kreitzer, Matthew A; Swygart, David; Osborn, Meredith; Skinner, Blair; Heer, Chad; Kaufman, Ryan; Williams, Bethany; Shepherd, Lexi; Caringal, Hannah; Gongwer, Michael; Tchernookova, Boriana K; Malchow, Robert P

2017-12-01

Self-referencing H + -selective electrodes were used to measure extracellular H + fluxes from Müller (glial) cells isolated from the tiger salamander retina. A novel chamber enabled stable recordings using H + -selective microelectrodes in a self-referencing format using bicarbonate-based buffer solutions. A small basal H + flux was observed from the end foot region of quiescent cells bathed in 24 mM bicarbonate-based solutions, and increasing extracellular potassium induced a dose-dependent increase in H + flux. Barium at 6 mM also increased H + flux. Potassium-induced extracellular acidifications were abolished when bicarbonate was replaced by 1 mM HEPES. The carbonic anhydrase antagonist benzolamide potentiated the potassium-induced extracellular acidification, while 300 μM DIDS, 300 μM SITS, and 30 μM S0859 significantly reduced the response. Potassium-induced extracellular acidifications persisted in solutions lacking extracellular calcium, although potassium-induced changes in intracellular calcium monitored with Oregon Green were abolished. Exchange of external sodium with choline also eliminated the potassium-induced extracellular acidification. Removal of extracellular sodium by itself induced a transient alkalinization, and replacement of sodium induced a transient acidification, both of which were blocked by 300 μM DIDS. Recordings at the apical portion of the cell showed smaller potassium-induced extracellular H + fluxes, and removal of the end foot region further decreased the H + flux, suggesting that the end foot was the major source of acidifications. These studies demonstrate that self-referencing H + -selective electrodes can be used to monitor H + fluxes from retinal Müller cells in bicarbonate-based solutions and confirm the presence of a sodium-coupled bicarbonate transporter, the activity of which is largely restricted to the end foot of the cell. NEW & NOTEWORTHY The present study uses self-referencing H + -selective electrodes for the first time to measure H + fluxes from Müller (glial) cells isolated from tiger salamander retina. These studies demonstrate bicarbonate transport as a potent regulator of extracellular levels of acidity around Müller cells and point toward a need for further studies aimed at addressing how such glial cell pH regulatory mechanisms may shape neuronal signaling. Copyright © 2017 the American Physiological Society.

The antioxidant capacity of erythrocyte concentrates is increased during the first week of storage and correlated with the uric acid level.

PubMed

Bardyn, M; Maye, S; Lesch, A; Delobel, J; Tissot, J-D; Cortés-Salazar, F; Tacchini, P; Lion, N; Girault, H H; Prudent, M

2017-10-01

Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage. Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43 days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour. The AOP of total ECs and their extracellular samples attained a maximum after 1 week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors. The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied. © 2017 International Society of Blood Transfusion.

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

PubMed

Fukuuchi, T; Iyama, N; Yamaoka, N; Kaneko, K

2018-04-13

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Physical effects at the cellular level under altered gravity conditions

NASA Technical Reports Server (NTRS)

Todd, Paul

1992-01-01

Several modifications of differentiated functions of animal cells cultivated in vitro have been reported when cultures have been exposed to increased or decreased inertial acceleration fields by centrifugation, clinorotation, and orbital space flight. Variables modified by clinorotation conditions include inertial acceleration, convection, hydrostatic pressure, sedimentation, and shear stress, which also affect transport processes in the extracellular chemical environment. Autocrine, paracrine and endocrine substances, to which cells are responsive via specific receptors, are usually transported in vitro (and possibly in certain embryos) by convection and in vivo by a circulatory system or ciliary action. Increased inertial acceleration increases convective flow, while microgravity nearly abolishes it. In the latter case the extracellular transport of macromolecules is governed by diffusion. By making certain assumptions it is possible to calculate the Peclet number, the ratio of convective transport to diffusive transport. Some, but not all, responses of cells in vitro to modified inertial environments could be manifestations of modified extracellular convective flow.

Neutrophil extracellular traps promote deep vein thrombosis in mice

PubMed Central

Brill, A.; Fuchs, T.A.; Savchenko, A.S.; Thomas, G.M.; Martinod, K.; De Meyer, S.F.; Bhandari, A.A.; Wagner, D.D.

2011-01-01

Summary Background Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be pro-thrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). Objective To explore the source and role of extracellular chromatin in DVT. Methods We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). Results We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared to sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs’ structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Conclusions Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development. PMID:22044575

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2*

PubMed Central

Brechbuhl, Heather M.; Gould, Neal; Kachadourian, Remy; Riekhof, Wayne R.; Voelker, Dennis R.; Day, Brian J.

2010-01-01

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2′,5′-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2′,5′-dihydroxychalcone (2′,5′-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 μm), indicating competitive inhibition. In contrast, 2′,5′-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter. PMID:20332504

Glutathione transport is a unique function of the ATP-binding cassette protein ABCG2.

PubMed

Brechbuhl, Heather M; Gould, Neal; Kachadourian, Remy; Riekhof, Wayne R; Voelker, Dennis R; Day, Brian J

2010-05-28

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer

PubMed Central

Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

2016-01-01

Background Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM) remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC), promoting a microenvironment favorable for tumor growth. Methods Serum samples obtained from 79 subjects [26 lean (LN) and 53 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC). Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN), chitinase-3-like protein 1 (YKL-40), tenascin C (TNC) and lipocalin-2 (LCN-2) were determined by ELISA. Results Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01) and colon cancer (P<0.05). LCN-2 levels were affected by obesity (P<0.05), but no differences were detected regarding the presence or not of CC. A positive association (P<0.05) with different inflammatory markers was also detected. Conclusions To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics. PMID:27612200

Similar increases in extracellular lactic acid in the limbic system during epileptic and/or olfactory stimulation.

PubMed

Fornai, F; Bassi, L; Gesi, M; Giorgi, F S; Guerrini, R; Bonaccorsi, I; Alessandrì, M G

2000-01-01

Previous studies have shown that physiological stimulation of brain activity increases anaerobic glucose consumption, both in humans and in experimental animals. To investigate this phenomenon further, we measured extracellular lactate levels within different rat brain regions, using microdialysis. Experiments were performed comparing the effects of natural, physiological olfactory stimulation of the limbic system with experimental limbic seizures. Olfactory stimulation was carried out by using different odors (i.e. both conventional odors: 2-isobutyl-3-methoxypyrazine, green pepper essence; thymol; and 2-sec-butylthiazoline, a sexual pheromone). Limbic seizures were either induced by systemic injection of pilocarpine (200-400 mg/kg) or focally elicited by microinfusions of chemoconvulsants (bicuculline 118 pmol and cychlothiazide 1.2 nmol) within the anterior piriform cortex. Seizures induced by systemic pilocarpine tripled lactic acid within the hippocampus, whereas limbic seizures elicited by focal microinfusion of chemoconvulsants within the piriform cortex produced a less pronounced increase in extracellular lactic acid. Increases in extracellular lactate occurring during olfactory stimulation with the sexual pheromone (three times the baseline levels) were non-significantly different from those occurring after systemic pilocarpine. Increases in lactic acid following natural olfactory stimulation were abolished both by olfactory bulbectomy and by the focal microinfusion of tetrodotoxin, while they were significantly attenuated by the local application of the N-methyl-D-aspartate antagonist AP-5. Increases in hippocampal lactate induced by short-lasting stimuli (olfactory stimulation or microinfusion of subthreshold doses of chemoconvulsants, bicuculline 30 pmol) were reproducible after a short delay (1 h) and cumulated when applied sequentially. In contrast, limbic status epilepticus led to a long-lasting refractoriness to additional lactate-raising stimuli and there was no further increase in lactate levels when the olfactory stimulation was produced during status epilepticus. Increases in lactic acid following olfactory stimulation occurred with site specificity in the rhinencephalon (hippocampus, piriform and entorhinal cortex) but not in the dorsal striatum. Site specificity crucially relied on the quality of the stimulus. For instance, other natural stimuli (i.e. tail pinch) produced a similar increase in extracellular lactate in all brain areas under investigation. The major conclusion of this work is that the presentation of an odor known to be a rat pheromone results in lactate production as great as that induced by the systemic convulsant pylocarpine (maximum: 2.286+/-0.195 mM and 1.803+/-0.108 mM, respectively). This supports the notion that the great magnitude of lactate production known to accompany seizures can result from the intensified neural activity per se ("aerobic gycolysis"), not merely from local anoxia or other pathological changes.

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

PubMed

Chanalaris, Anastasios; Doherty, Christine; Marsden, Brian D; Bambridge, Gabriel; Wren, Stephen P; Nagase, Hideaki; Troeberg, Linda

2017-10-01

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C 51 H 40 N 6 O 23 S 6 ) bound to TIMP-3 with a K D value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonyl bis (imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis -1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis. Copyright © 2017 by The Author(s).

Increase of extracellular glutamate concentration increases its oxidation and diminishes glucose oxidation in isolated mouse hippocampus: reversible by TFB-TBOA.

PubMed

Torres, Felipe Vasconcelos; Hansen, Fernanda; Locks-Coelho, Lucas Doridio

2013-08-01

Glutamate concentration at the synaptic level must be kept low in order to prevent excitotoxicity. Astrocytes play a key role in brain energetics, and also astrocytic glutamate transporters are responsible for the vast majority of glutamate uptake in CNS. Experiments with primary astrocytic cultures suggest that increased influx of glutamate cotransported with sodium at astrocytes favors its flux to the tricarboxylic acid cycle instead of the glutamate-glutamine cycle. Although metabolic coupling can be considered an emergent field of research with important recent discoveries, some basic aspects of glutamate metabolism still have not been characterized in brain tissue. Therefore, the aim of this study was to investigate whether the presence of extracellular glutamate is able to modulate the use of glutamate and glucose as energetic substrates. For this purpose, isolated hippocampi of mice were incubated with radiolabeled substrates, and CO2 radioactivity and extracellular lactate were measured. Our results point to a diminished oxidation of glucose with increasing extracellular glutamate concentration, glutamate presumably being the fuel, and might suggest that oxidation of glutamate could buffer excitotoxic conditions by high glutamate concentrations. In addition, these findings were reversed when glutamate uptake by astrocytes was impaired by the presence of (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA). Taken together, our findings argue against the lactate shuttle theory, because glutamate did not cause any detectable increase in extracellular lactate content (or, presumably, in glycolysis), because the glutamate is being used as fuel instead of going to glutamine and back to neurons. Copyright © 2013 Wiley Periodicals, Inc.

Fluoxetine impairs insulin secretion without modifying extracellular serotonin levels in MIN6 β-cells.

PubMed

Cataldo, L R; Cortés, V A; Mizgier, M L; Aranda, E; Mezzano, D; Olmos, P; Galgani, J E; Suazo, J; Santos, J L

2015-09-01

Pancreatic β-cells synthetize and store Serotonin (5-Hydroxytriptamine, 5HT) which is co-released with insulin. It has been proposed that extracellular 5HT binds to specific cell surface receptors and modulate insulin secretion. On the other hand, Selective Serotonin Reuptake Inhibitor (SSRI) fluoxetine seems to reduce Glucose-Stimulated Insulin Secretion (GSIS). However, it is unknown whether this effect results from changes in extracellular 5HT concentration owed to the blockade of 5HT transporter (SERT) or from non-5HT dependent actions. The aims of this work were: 1) to quantify extracellular 5HT levels and GSIS in β-cell lines, 2) to determine whether extracellular 5HT levels and GSIS are changed by fluoxetine or 5-Hydroxytryptophan (5HTP, the immediate 5HT biosynthetic precursor), and 3) to quantify the expression of Slc6a4 gene (encoding SERT) in β-cell lines in relation to other genes involved in 5HT system. β-cell lines MIN6 and RINm5f were subjected to GSIS protocols, after treatment with fluoxetine, 5HTP or 5HT. Insulin and 5HT were quantified by ELISA and HPLC, respectively. Relative mRNA expression was quantified by RT-qPCR. MIN6 β-cells secretes 5HT in response to glucose, showing a sharp increase in 5HT release when cells were preloaded with 5HTP. Treatment with 5HT or fluoxetine reduces GSIS. Fluoxetine fails to further increases 5HTP-induced elevation of secreted 5HT. MIN6 β-cells express both isoforms of Tryptophan Hydroxylase (Tph1 and Tph2), and have high expression levels of L-Dopa decarboxylase (Ddc), both enzymes involved in 5HT biosynthetic pathway, but do not express the 5HT transporters Slc6a4 or Slc6a3 (the Dopamine-5HT transporter) genes. The inhibitory effect of fluoxetine on β-cell glucose stimulated insulin secretion is not mediated by blockage of 5HT transporter through SERT. © Georg Thieme Verlag KG Stuttgart · New York.

Gamma-vinyl GABA increases nonvesicular release of GABA and glutamate in the nucleus accumbens in rats via action on anion channels and GABA transporters

PubMed Central

Peng, Xiao-Qing; Gardner, Eliot L.

2013-01-01

Rationale γ-Amino butyric acid (GABA) is a well-characterized inhibitory neurotransmitter in the central nervous system, which may also stimulate nonvesicular release of other neurotransmitters under certain conditions. We have recently reported that γ-vinyl GABA (GVG), an irreversible GABA transaminase inhibitor, elevates extracellular GABA but fails to alter dopamine release in the nucleus accumbens (NAc). Objectives Here, we investigated the mechanism(s) by which GVG elevates extracellular GABA levels and whether GVG also alters glutamate release in the NAc. Materials and methods In vivo microdialysis was used to simultaneously measure extracellular NAc GABA and glutamate before and after GVG administration in freely moving rats. Results Systemic administration of GVG or intra-NAc local perfusion of GVG significantly increased extracellular NAc GABA and glutamate. GVG-enhanced GABA was completely blocked by intra-NAc local perfusion of 5-nitro-2, 3-(phenylpropylamino)-benzoic acid (NPPB), a selective anion channel blocker and partially blocked by SKF89976A, a type 1 GABA transporter inhibitor. GVG-enhanced glutamate was completely blocked by NPPB or SKF89976A. Tetrodotoxin, a voltage-dependent Na+-channel blocker, failed to alter GVG-enhanced GABA and glutamate. Conclusions These data suggest that GVG-enhanced extracellular GABA and glutamate are mediated predominantly by the opening of anion channels and partially by the reversal of GABA transporters. Enhanced extracellular glutamate may functionally attenuate the pharmacological action of GABA and prevent enhanced GABA-induced excess inhibition. PMID:20033132

Characteristic of Extracellular Zn2+ Influx in the Middle-Aged Dentate Gyrus and Its Involvement in Attenuation of LTP.

PubMed

Takeda, Atsushi; Koike, Yuta; Osaw, Misa; Tamano, Haruna

2018-03-01

An increased influx of extracellular Zn 2+ into neurons is a cause of cognitive decline. The influx of extracellular Zn 2+ into dentate granule cells was compared between young and middle-aged rats because of vulnerability of the dentate gyrus to aging. The influx of extracellular Zn 2+ into dentate granule cells was increased in middle-aged rats after injection of AMPA and high K + into the dentate gyrus, but not in young rats. Simultaneously, high K + -induced attenuation of LTP was observed in middle-aged rats, but not in young rats. The attenuation was rescued by co-injection of CaEDTA, an extracellular Zn 2+ chelator. Intracellular Zn 2+ in dentate granule cells was also increased in middle-aged slices with high K + , in which the increase in extracellular Zn 2+ was the same as young slices with high K + , suggesting that ability of extracellular Zn 2+ influx into dentate granule cells is greater in middle-aged rats. Furthermore, extracellular zinc concentration in the hippocampus was increased age-dependently. The present study suggests that the influx of extracellular Zn 2+ into dentate granule cells is more readily increased in middle-aged rats and that its increase is a cause of age-related attenuation of LTP in the dentate gyrus.

Extracellular potassium changes in the rat neurohypophysis during activation of the magnocellular neurosecretory system.

PubMed Central

Leng, G; Shibuki, K

1987-01-01

1. Potassium-sensitive microelectrodes were used to measure extracellular [K+] in the isolated rat neurohypophysis maintained in vitro. Electrical stimulation of the neurohypophysial stalk (20 Hz 5 s) increased the inferred extracellular [K+] by 9.2 +/- 0.4 mM (mean +/- S.E. of mean; n = 21). 2. Veratridine (10 microM) enhanced the response to stalk stimulation, and at a higher concentration (50 microM) increased extracellular [K+] in the absence of stimulation. By contrast, tetrodotoxin (1 microM) blocked the [K+] increase completely and reversibly in each of five experiments, indicating that the increase was a consequence of action potential generation. 3. At the end of brief periods of stimulation, the raised extracellular [K+] returned to pre-stimulation levels within 30 s. In the presence of ouabain (100 microM), the recovery was slower: the half-decay time was extended by 150-300% in each of three experiments. 4. Replacement of calcium in the medium with cobalt, cadmium or magnesium reduced the amplitude of the [K+] increase by 26-30%, indicating that the [K+] increase was largely independent of events subsequent to evoked release of hormone and/or transmitters. 5. Potassium-sensitive microelectrodes were placed in the neurohypophysis of rats anaesthetized with urethane. Electrical stimulation of the pituitary stalk (50 Hz, 5 s) produced transient voltage increases of 7.6 +/- 0.9 mV (mean +/- S.E. of mean of seven experiments). These voltage increases were similar in magnitude to the response of the electrodes to the addition of 7.6 +/- 1.0 mM-K+ to rat plasma. 6. In seven lactating rats, the suckling of a litter of hungry pups evoked periodic reflex milk ejections, as detected by increases in intramammary pressure. Potassium-sensitive microelectrodes in the neurohypophysis recorded transient voltage increases prior to each milk ejection (0.4-5.5 mV). Each increase preceded an increase in intramammary pressure by 12-30 s. 7. Thus synchronized high-frequency activation of magnocellular neurones can produce large changes in extracellular [K+]. The implications of these findings for stimulus-secretion coupling in the neurohypophysis are discussed in the light of previous reports that hormone release from the neurohypophysis is highly dependent on the frequency and pattern of electrical stimulation. PMID:2451734

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV.

PubMed

Verónica Donoso, M; Hernández, Felipe; Villalón, Tania; Acuña-Castillo, Claudio; Pablo Huidobro-Toro, J

2018-06-01

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca 2+ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1 -/- rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells.

PubMed Central

Riser, B. L.; Cortes, P.; Heilig, C.; Grondin, J.; Ladson-Wofford, S.; Patterson, D.; Narins, R. G.

1996-01-01

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension. Images Figure 1 Figure 3 PMID:8669477

Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons

PubMed Central

Borges, Gisela Patrícia; Micó, Juan Antonio; Neto, Fani Lourença

2015-01-01

Background: The corticotropin-releasing factor is a stress-related neuropeptide that modulates locus coeruleus activity. As locus coeruleus has been involved in pain and stress-related patologies, we tested whether the pain-induced anxiety is a result of the corticotropin-releasing factor released in the locus coeruleus. Methods: Complete Freund’s adjuvant-induced monoarthritis was used as inflammatory chronic pain model. α-Helical corticotropin-releasing factor receptor antagonist was microinjected into the contralateral locus coeruleus of 4-week-old monoarthritic animals. The nociceptive and anxiety-like behaviors, as well as phosphorylated extracellular signal-regulated kinases 1/2 and corticotropin-releasing factor receptors expression, were quantified in the paraventricular nucleus and locus coeruleus. Results: Monoarthritic rats manifested anxiety and increased phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus and paraventricular nucleus, although the expression of corticotropin-releasing factor receptors was unaltered. α-Helical corticotropin-releasing factor antagonist administration reversed both the anxiogenic-like behavior and the phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus. Conclusions: Pain-induced anxiety is mediated by corticotropin-releasing factor neurotransmission in the locus coeruleus through extracellular signal-regulated kinases 1/2 signaling cascade. PMID:25716783

Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90.

PubMed

Zhang, Guohua; Liu, Zhelong; Ding, Hui; Zhou, Yong; Doan, Hoang Anh; Sin, Ka Wai Thomas; Zhu, Zhiren J; Flores, Rene; Wen, Yefei; Gong, Xing; Liu, Qingyun; Li, Yi-Ping

2017-09-19

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor's capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.Cachexia affects many cancer patients causing weight loss and increasing mortality. Here, the authors identify extracellular Hsp70 and Hsp90, either in soluble form or secreted as part of exosomes from tumor cells, to be responsible for tumor induction of cachexia.

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis.

PubMed

Rex, A; Bert, B; Fink, H; Voigt, J-P

2009-10-19

Neuronal activity is tightly coupled with brain energy metabolism; and glucose is an important energy substrate for neurons. The present in vivo microdialysis study was aimed at investigating changes in extracellular glucose concentrations in the rat ventral hippocampus due to exposure to the elevated plus maze. Determination of basal hippocampal glucose and lactate/pyruvate ratio in male Wistar rats was conducted in the home cage using in vivo microdialysis. Rats were exposed to the elevated plus maze, a rodent model of anxiety-related behaviour, or to unspecific stress induced by white noise (95dB) as a control condition. Basal hippocampal levels of glucose, as determined by zero-net-flux, and the basal lactate/pyruvate ratio were 1.49+/-0.05mmol/l and 13.8+/-1.1, respectively. In rats without manipulation, glucose levels remained constant throughout the experiment (120min). By contrast, exposure to the elevated plus maze led to a temporary decline in hippocampal glucose (-33.2+/-4.4%) which returned to baseline level in the home cage. White noise caused only a non-significant decrease in extracellular glucose level (-9.3+/-3.5%). In all groups, the lactate/pyruvate ratio remained unchanged by the experimental procedures. Our microdialysis study demonstrates that exposure to the elevated plus maze induces a transient decrease in extracellular hippocampal glucose concentration. In contrast, an unspecific stimulus did not change hippocampal glucose. The latter suggests that only specific behavioural stimuli increase hippocampal glucose utilization in the ventral hippocampus.

ROS production, intracellular HSP70 levels and their relationship in human neutrophils: effects of age.

PubMed

Kovalenko, Elena I; Boyko, Anna A; Semenkov, Victor F; Lutsenko, Gennady V; Grechikhina, Maria V; Kanevskiy, Leonid M; Azhikina, Tatyana L; Telford, William G; Sapozhnikov, Alexander M

2014-12-15

ROS production and intracellular HSP70 levels were measured in human neutrophils for three age groups: young (20-59 years), elders (60-89 years) and nonagenarians (90 years and older). Elders showed higher levels of spontaneous intracellular ROS content compared with young and nonagenarian groups, which had similar intracellular ROS levels. Zymosan-induced (non-spontaneous) extracellular ROS levels were also similar for young and nonagenarians but were lower in elders. However, spontaneous extracellular ROS production increased continuously with age. Correlation analysis revealed positive relationships between HSP70 levels and zymosan-stimulated ROS production in the elder group. This was consistent with a promoting role for HSP70 in ROS-associated neutrophils response to pathogens. No positive correlation between ROS production and intracellular HSP70 levels was found for groups of young people and nonagenarians. In contrast, significant negative correlations of some ROS and HSP70 characteriscics were found for neutrophils from young people and nonagenarians. The observed difference in ROS and HSP70 correlations in elders and nonagenarians might be associated with an increased risk of mortality in older individuals less than 90 years old.

Uric acid is released in the brain during seizure activity and increases severity of seizures in a mouse model for acute limbic seizures.

PubMed

Thyrion, Lisa; Raedt, Robrecht; Portelli, Jeanelle; Van Loo, Pieter; Wadman, Wytse J; Glorieux, Griet; Lambrecht, Bart N; Janssens, Sophie; Vonck, Kristl; Boon, Paul

2016-03-01

Recent evidence points at an important role of endogenous cell-damage induced pro-inflammatory molecules in the generation of epileptic seizures. Uric acid, under the form of monosodium urate crystals, has shown to have pro-inflammatory properties in the body, but less is known about its role in seizure generation. This study aimed to unravel the contribution of uric acid to seizure generation in a mouse model for acute limbic seizures. We measured extracellular levels of uric acid in the brain and modulated them using complementary pharmacological and genetic tools. Local extracellular uric acid levels increased three to four times during acute limbic seizures and peaked between 50 and 100 min after kainic acid infusion. Manipulating uric acid levels through administration of allopurinol or knock-out of urate oxidase significantly altered the number of generalized seizures, decreasing and increasing them by a twofold respectively. Taken together, our results consistently show that uric acid is released during limbic seizures and suggest that uric acid facilitates seizure generalization. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential effects of pyrethroid insecticides on extracellular dopamine in the striatum of freely moving rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mubarak Hossain, Muhammad; Suzuki, Tadahiko; United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193

2006-11-15

In order to obtain a more complete understanding of pyrethroid neurotoxicity, effects of the pyrethroid insecticides, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II) on extracellular levels of dopamine (DA) and its metabolites in the striatum of conscious rats were studied by in vivo microdialysis. Rats were treated i.p. with pyrethroids or vehicle. Allethrin had a dual effect on DA release. The increase in the extracellular level of striatal DA by 10 mg/kg allethrin reached a maximum of 178% of baseline but 20 and 60 mg/kg inhibited DA release to 63% and 52% of baseline with a peakmore » effect at 60-80 min after injection. Cyhalothrin 10, 20 and 60 mg/kg inhibited DA release to 65%, 56% and 45% of basal release, respectively, with a peak time of inhibition 40-80 min past injection. Deltamethrin (10 and 20 mg/kg) increased DA release to maximum of 187% and 252% of basal release whereas 60 mg/kg first reduced the efflux for 40 min to 50% of basal release and then increased the efflux to a maximum of 344% of basal release with a peak time of 120 min. Local infusion of 1 {mu}M tetrodotoxin, a Na{sup +} blocker through the dialysis probe completely prevented the effect of allethrin (10 and 60 mg/kg), cyhalothrin (60 mg/kg) and deltamethrin (20 mg/kg) on DA release but only partially blocked the effects of 60 mg/kg deltamethrin. The effect of deltamethrin (60 mg/kg) on DA release was completely prevented by local infusion of 10 {mu}M nimodipine, an L-type Ca{sup ++} channel blocker. All three pyrethroids did not alter the extracellular levels of DOPAC, 3-MT and HVA except that 20 and 60 mg/kg of allethrin and cyhalothrin increased 3-MT levels. Effect of the pyrethroids on synaptosomal DA uptake was also examined. The DA uptake was decreased in rats exposed to 60 mg/kg of allethrin and cyhalothrin but was increased in rats exposed to 60 mg/kg of deltamethrin. Our results demonstrate that striatal DA release and DA uptake are differentially affected by type I and the two type II pyrethroids indicating that dopaminergic circuitry, striatal DA in particular, may be a pyrethroid target and that pyrethroids may be acting on striatal DA by multiple mechanisms.« less

In vivo neurobiological effects of ibogaine and its O-desmethyl metabolite, 12-hydroxyibogamine (noribogaine), in rats.

PubMed

Baumann, M H; Rothman, R B; Pablo, J P; Mash, D C

2001-05-01

Ibogaine is a naturally occurring compound with purported antiaddictive properties. When administered to primates, ibogaine is rapidly o-demethylated to form the metabolite 12-hydroxyibogamine (noribogaine). Peak blood levels of noribogaine exceed those of ibogaine, and noribogaine persists in the bloodstream for at least 1 day. Very few studies have systematically evaluated the neurobiological effects of noribogaine in vivo. In the present series of experiments, we compared the effects of i.v. administration of ibogaine and noribogaine (1 and 10 mg/kg) on motor behaviors, stress hormones, and extracellular levels of dopamine (DA) and serotonin (5-HT) in the nucleus accumbens of male rats. Ibogaine caused dose-related increases in tremors, whereas noribogaine did not. Both ibogaine and noribogaine produced significant elevations in plasma corticosterone and prolactin, but ibogaine was a more potent stimulator of corticosterone secretion. Neither drug altered extracellular DA levels in the nucleus accumbens. However, both drugs increased extracellular 5-HT levels, and noribogaine was more potent in this respect. Results from in vitro experiments indicated that ibogaine and noribogaine interact with 5-HT transporters to inhibit 5-HT uptake. The present findings demonstrate that noribogaine is biologically active and undoubtedly contributes to the in vivo pharmacological profile of ibogaine in rats. Noribogaine is approximately 10 times more potent than ibogaine as an indirect 5-HT agonist. More importantly, noribogaine appears less apt to produce the adverse effects associated with ibogaine, indicating the metabolite may be a safer alternative for medication development.

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins. PMID:24752318

Tumor cell-driven extracellular matrix remodeling drives haptotaxis during metastatic progression

PubMed Central

Oudin, Madeleine J.; Jonas, Oliver; Kosciuk, Tatsiana; Broye, Liliane C.; Guido, Bruna C.; Wyckoff, Jeff; Riquelme, Daisy; Lamar, John M.; Asokan, Sreeja B.; Whittaker, Charlie; Ma, Duanduan; Langer, Robert; Cima, Michael J.; Wisinski, Kari B.; Hynes, Richard O.; Lauffenburger, Douglas A.; Keely, Patricia J.; Bear, James E.; Gertler, Frank B.

2016-01-01

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration. PMID:26811325

Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

PubMed

Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

2015-09-01

Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg(2+) as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. © 2015 American Society of Plant Biologists. All Rights Reserved.

Increased L-DOPA-derived dopamine following selective MAO-A or-B inhibition in rat striatum depleted of dopaminergic and serotonergic innervation

PubMed Central

Sader-Mazbar, O; Loboda, Y; Rabey, M J; Finberg, J P M

2013-01-01

Background and Purpose Selective MAO type B (MAO-B) inhibitors are effective in potentiation of the clinical effect of L-DOPA in Parkinson's disease, but dopamine (DA) is deaminated mainly by MAO type A (MAO-A) in rat brain. We sought to clarify the roles of MAO-A and MAO-B in deamination of DA formed from exogenous L-DOPA in rat striatum depleted of dopaminergic, or both dopaminergic and serotonergic innervations. We also studied the effect of organic cation transporter-3 (OCT-3) inhibition by decinium-22 on extracellular DA levels following L-DOPA. Experimental Approach Striatal dopaminergic and/or serotonergic neuronal innervations were lesioned by 6-hydroxydopamine or 5,7-dihydroxytryptamine respectively. Microdialysate DA levels after systemic L-DOPA were measured after inhibition of MAO-A or MAO-B by clorgyline or rasagiline respectively. MAO subtype localization in the striatum was determined by immunofluorescence. Key Results Rasagiline increased DA extracellular levels following L-DOPA to a greater extent in double-than in single-lesioned rats (2.8-and 1.8-fold increase, respectively, relative to saline treatment); however, clorgyline elevated DA levels in both models over 10-fold. MAO-A was strongly expressed in medium spiny neurons (MSNs) in intact and lesioned striata, while MAO-B was localized in glia and to a small extent in MSNs. Inhibition of OCT-3 increased DA levels in the double-more than the single-lesion animals. Conclusions and Implications In striatum devoid of dopaminergic and serotonergic inputs, most deamination of L-DOPA-derived DA is mediated by MAO-A in MSN and a smaller amount by MAO-B in both MSN and glia. OCT-3 plays a significant role in uptake of DA from extracellular space. Inhibitors of OCT-3 are potential future targets for anti-Parkinsonian treatments. PMID:23992249

Involvement of amygdalar extracellular zinc in rat behavior for passive avoidance.

PubMed

Takeda, Atsushi; Minami, Akira; Yamaide, Rie; Oku, Naoto

2004-03-25

On the basis of the evidence that zinc is released from glutamatergic neuron terminals in the amygdala, the effect of chelation of amygdalar extracellular zinc on glutamate release from the neuron terminals was studied by using in vivo microdialysis. When the amygdala was perfused with 100 microM CaEDTA to chelate extracellular zinc, glutamate concentration in the perfusate was decreased significantly, whereas that tended to be increased by perfusion with 100 microM ZnEDTA as a control. The effect of CaEDTA on extracellular glutamate levels was different between the amygdala and hippocampus, implying that modulation of glutamate signaling by zinc is different between them. To evaluate chelation of zinc in rat behavior, perfusion of the amygdala with CaEDTA was started 40 min before behavioral test for passive avoidance. The behavior for passive avoidance was impaired during perfusion with CaEDTA. On the other hand, the behavior during perfusion with ZnEDTA was more rapidly developed than that with vehicle only. These results suggest that amygdalar extracellular zinc is involved in the behavior for passive avoidance.

Brain extracellular glucose assessed by voltammetry throughout the rat sleep-wake cycle.

PubMed

Netchiporouk, L; Shram, N; Salvert, D; Cespuglio, R

2001-04-01

In the present study, cortical extracellular levels of glucose were monitored for the first time throughout the sleep-wake states of the freely moving rat. For this purpose, polygraphic recordings (electroencephalogram of the fronto-occipital cortices and electromyogram of the neck muscles) were achieved in combination with differential normal pulse voltammetry (DNPV) using a specific glucose sensor. Data obtained reveal that the basal extracellular glucose concentration in the conscious rat is 0.59 +/- 0.3 m M while under chloral hydrate anaesthesia (0.4 g/kg, i.p.) it increases up to 180% of its basal concentration. Regarding the sleep-wake cycle, the existence of spontaneous significant variations in the mean glucose level during slow-wave sleep (SWS = +13%) and paradoxical sleep (PS = -11%) compared with the waking state (100%) is also reported. It is to be noticed that during long periods of active waking, glucose level tends towards a decrease that becomes significant after 15 min (active waking = -32%). On the contrary, during long episodes of slow-wave sleep, it tends towards an increase which becomes significant after 12 min (SWS = +28%). It is suggested that voltammetric techniques using enzymatic biosensors are useful tools allowing direct glucose measurements in the freely moving animal. On the whole, paradoxical sleep is pointed out as a state highly dependent on the availability of energy and slow-wave sleep as a period of energy saving.

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

PubMed

Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

2008-07-01

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate. 2008 Wiley-Liss, Inc.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

PubMed

Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, María Angeles; Sabater-Jara, Ana Belén

2018-07-01

Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight -1 ) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Behavior-associated and post-consumption glucose entry into the nucleus accumbens extracellular space during glucose free-drinking in trained rats

PubMed Central

Wakabayashi, Ken T.; Kiyatkin, Eugene A.

2015-01-01

Glucose is the primary energetic substrate for the metabolic activity of brain cells and its proper delivery from the arterial blood is essential for neural activity and normal brain functions. Glucose is also a unique natural reinforcer, supporting glucose-drinking behavior without food or water deprivation. While it is known that glucose enters brain tissue via gradient-dependent facilitated diffusion, it remains unclear how glucose levels are changed during natural behavior and whether the direct central action of ingested glucose can be involved in regulating glucose-drinking behavior. Here, we used glucose biosensors with high-speed amperometry to examine the pattern of phasic and tonic changes in extracellular glucose in the nucleus accumbens (NAc) during unrestricted glucose-drinking in well-trained rats. We found that the drinking behavior is highly cyclic and is associated with relatively large and prolonged increases in extracellular glucose levels. These increases had two distinct components: a highly phasic but relatively small behavior-related rise and a larger tonic elevation that results from the arrival of consumed glucose into the brain’s extracellular space. The large post-ingestion increases in NAc glucose began minutes after the cessation of drinking and were consistently associated with periods of non-drinking, suggesting that the central action of ingested glucose could inhibit drinking behavior by inducing a pause in activity between repeated drinking bouts. Finally, the difference in NAc glucose responses found between active, behavior-mediated and passive glucose delivery via an intra-gastric catheter confirms that motivated behavior is also associated with metabolic glucose use by brain cells. PMID:26190984

Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

PubMed

Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

2014-04-01

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2

PubMed Central

Ohse, Takamoto; Krofft, Ron D.; Wu, Jimmy S.; Eddy, Allison A.; Pippin, Jeffrey W.; Shankland, Stuart J.

2012-01-01

Background. The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. Methods. Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. Results. PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P < 0.05 versus controls). Cultured immortalized PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced apoptosis in cultured PECs, while a forced decrease of p-ERK1/2 through inhibition of MEK 1/2 significantly increased albumin-induced PEC apoptosis. Conclusions. A normal role of PECs is to take up filtered albumin. However, this is increased in proteinuric glomerular diseases, leading to apoptosis through changes in ERK1/2. PMID:21896500

[GABA-NO interaction in the N. Accumbens during danger-induced inhibition of exploratory behavior].

PubMed

Saul'skaia, N V; Terekhova, E A

2013-01-01

In Sprague-Dawley rats by means of in vivo microdialysis combined with HPLC analysis, it was shown that presentation to rats during exploratory activity of a tone previously pared with footshock inhibited the exploration and prevented the exploration-induced increase in extracellular levels of citrulline (an NO co-product) in the medial n. accumbens. Intra-accumbal infusions of 20 μM bicuculline, a GABA(A)-receptor antagonist, firstly, partially restored the exploration-induced increase of extracellular citrulline levels in this brain area, which was inhibited by presentation of the tone, previously paired with foot-shock and, secondly, prevented the inhibition of exploratory behavior produced by this sound signal of danger. The data obtained indicate for the first time that signals of danger inhibit exploratory behavior and exploration-induced activation of the accumbal nitrergic system via GABA(A)-receptor mechanisms.

[Vesicular and nonvesicular glutamate release in the nucleus accumbens during a forced switch in behavioral strategy].

PubMed

Saul'skaia, N B; Mikhaĭlova, M O

2004-01-01

By means of in vivo microdialysis combined with HPLC analysis, we have shown that glutamate extracellular level in the rat n. accumbens increases during a forced switch in behavioral strategy. When infused in the n. accumbens, a Na+ channel blocker tetrodotoxin (TTX, 1 microM) completely prevents this increase whereas a potent cystine/glutamate exchanger blocker (S)-4-carboxyphenylglycine ((S)-4-CPG, 5 microM) has no effect. In contrast, TT (1 microM), infused in the n. accumbens, fails to significantly alter basal level of extracellular glutamate in this region whereas (S)-4-CPG (5 microM) produced a significant decrease. Our data suggest that basal and factional glutamate releases in the n. accumbens are differently regulated. The source of basal glutamate release is a non-vesicular release via cystine/glutamate exchanger. Functional glutamate release observed during a forced switch in behavioral strategy derives from vesicular synaptic pool.

Agmatine reduces extracellular glutamate during pentylenetetrazole-induced seizures in rat brain: A potential mechanism for the anticonvulsive effects

PubMed Central

Feng, Yangzheng; LeBlanc, Michael H.; Regunathan, Soundar

2010-01-01

Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that reached brain after peripheral injection. After i.p. injection of agmatine (50 mg/kg), increase of agmatine in rat cortex and hippocampus was observed in 15 min with levels returning to baseline in one hour. Rats, naïve and implanted with microdialysis cannula into the cortex, were administered PTZ (60 mg/kg, i.p.) with prior injection of agmatine (100 mg/kg, i.p.) or saline. Seizure grades were recorded and microdialysis samples were collected every 15 min for 75 min. Agmatine pre-treatment significantly reduced the seizure grade and increased the onset time. The levels of extracellular glutamate in frontal cortex rose two- to three-fold after PTZ injection and agmatine significantly inhibited this increase. In conclusion, the present data suggest that the anticonvulsant activity of agmatine, in part, could be related to the inhibition glutamate release. PMID:16125317

Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tada, Hiroyuki; Nemoto, Eiji, E-mail: e-nemoto@umin.ac.jp; Kanaya, Sousuke

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stabilitymore » of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.« less

Analysis of the inter- and extracellular formation of platinum nanoparticles by Fusarium oxysporum f. sp. lycopersici using response surface methodology

NASA Astrophysics Data System (ADS)

Riddin, T. L.; Gericke, M.; Whiteley, C. G.

2006-07-01

Fusarium oxysporum fungal strain was screened and found to be successful for the inter- and extracellular production of platinum nanoparticles. Nanoparticle formation was visually observed, over time, by the colour of the extracellular solution and/or the fungal biomass turning from yellow to dark brown, and their concentration was determined from the amount of residual hexachloroplatinic acid measured from a standard curve at 456 nm. The extracellular nanoparticles were characterized by transmission electron microscopy. Nanoparticles of varying size (10-100 nm) and shape (hexagons, pentagons, circles, squares, rectangles) were produced at both extracellular and intercellular levels by the Fusarium oxysporum. The particles precipitate out of solution and bioaccumulate by nucleation either intercellularly, on the cell wall/membrane, or extracellularly in the surrounding medium. The importance of pH, temperature and hexachloroplatinic acid (H2PtCl6) concentration in nanoparticle formation was examined through the use of a statistical response surface methodology. Only the extracellular production of nanoparticles proved to be statistically significant, with a concentration yield of 4.85 mg l-1 estimated by a first-order regression model. From a second-order polynomial regression, the predicted yield of nanoparticles increased to 5.66 mg l-1 and, after a backward step, regression gave a final model with a yield of 6.59 mg l-1.

NUTRITION AND METABOLISM OF MARINE BACTERIA1

PubMed Central

Takacs, Frank P.; Matula, Tibor I.; MacLeod, Robert A.

1964-01-01

Takacs, Frank P. (McGill University, Montreal, Quebec, Canada), Tibor I. Matula, and Robert A. MacLeod. Nutrition and metabolism of marine bacteria. XIII. Intracellular concentrations of sodium and potassium ions in a marine pseudomonad. J. Bacteriol. 87:510–518. 1964.—Washed cells of a marine pseudomonad were suspended in buffered salt solutions containing, in addition to MgSO4, NaCl and KCl at various concentrations. The cells were centrifuged from the medium and analyzed for Na+ and K+. Inulin and C14-carboxypolyglucose were employed to estimate the volume of extracellular fluid associated with the packed cells. Intracellular Na+ and K+ concentrations were determined by correcting for the amount of Na+ and K+ in the extracellular fluid. At all levels of Na+ in the suspending medium (0 to 1 m), the intracellular and extracellular Na+ concentrations within the limits of experimental error were the same. The intracellular K+ concentrations were approximately double the extracellular concentrations at the two levels of K+ tested (0.01 and 0.15 m) and were not influenced by the amount of Na+ present. Intracellular and extracellular Cl− concentrations were the same at the one level of Cl− examined. The intracellular fluid volume varied with the NaCl or KCl concentration of the suspending medium, being greatest in the absence of added salts, decreasing to a minimum at 0.3 m salt, and then increasing slightly at higher salt concentrations. Most of the intracellular Na+ could be removed by washing with solutions of MgSO4 or sucrose, but a small amount [10 to 15 μmoles/g (dry weight)] remained bound to the cells. PMID:14129666

Extracellular Superoxide Dismutase Deficiency Exacerbates Pressure Overload–Induced Left Ventricular Hypertrophy and Dysfunction

PubMed Central

Lu, Zhongbing; Xu, Xin; Hu, Xinli; Zhu, Guangshuo; Zhang, Ping; van Deel, Elza D.; French, Joel P.; Fassett, John T.; Oury, Tim D.; Bache, Robert J.; Chen, Yingjie

2008-01-01

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)–induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload. PMID:17998475

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

PubMed Central

Gonçalves, A. P.; Monteiro, João; Lucchi, Chiara; Kowbel, David J.; Cordeiro, J. M.; Correia-de-Sá, Paulo; Rigden, Daniel J.; Glass, N. L.; Videira, Arnaldo

2014-01-01

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. PMID:28357255

Microfluidic Approaches for Isolation, Detection, and Characterization of Extracellular Vesicles: Current Status and Future Directions

PubMed Central

Gholizadeh, Shima; Draz, Mohamed; Zarghooni, Maryam; Nezhad, Amir Sanati; Ghavami, Saeid; Shafiee, Hadi; Akbari, Mohsen

2017-01-01

Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complication and inflammatory disorders. Despite increasing scientific and clinical interest in this field, at the time of writing there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs. PMID:28088752

Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

PubMed Central

Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

2017-01-01

Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

New insights into the early steps of oil body mobilization during pollen germination

PubMed Central

Castro, Antonio Jesús

2013-01-01

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids. PMID:23132905

Regulation of Cellular Calcium in Vestibular Supporting Cells by Otopetrin 1

PubMed Central

Kim, Euysoo; Hyrc, Krzysztof L.; Speck, Judith; Lundberg, Yunxia W.; Salles, Felipe T.; Kachar, Bechara; Goldberg, Mark P.; Warchol, Mark E.

2010-01-01

Otopetrin 1 (OTOP1) is a multitransmembrane domain protein, which is essential for mineralization of otoconia, the calcium carbonate biominerals required for vestibular function, and the normal sensation of gravity. The mechanism driving mineralization of otoconia is poorly understood, but it has been proposed that supporting cells and a mechanism to maintain high concentrations of calcium are critical. Using Otop1 knockout mice and a utricular epithelial organ culture system, we show that OTOP1 is expressed at the apex of supporting cells and functions to increase cytosolic calcium in response to purinergic agonists, such as adenosine 5′-triphosphate (ATP). This is achieved by blocking mobilization of calcium from intracellular stores in an extracellular calcium-dependent manner and by mediating influx of extracellular calcium. These data support a model in which OTOP1 acts as a sensor of the extracellular calcium concentration near supporting cells and responds to ATP in the endolymph to increase intracellular calcium levels during otoconia mineralization. PMID:20554841

Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

PubMed Central

Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-01-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

PubMed

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Apamin increases 5-HT cell firing in raphe dorsalis and extracellular 5-HT levels in amygdala: a concomitant in vivo study in anesthetized rats.

PubMed

Crespi, F

2009-07-24

The family of calcium-activated slow-potassium (SK) channels comprises 3 members, the SK1, SK2 and SK3 channels, all expressed in neurons, known to mediate the slow-afterhyperpolarization occurring after action potentials. In rats, the SK2 and SK3 channels are expressed in the ascending monoaminergic systems, in particular in the serotonin (5-HT) neurons of the raphe dorsalis nucleus (RDN). In mammals the amygdala, a limbic structure involved in the control of emotion and mood, receives 5-HT-containing projections originating in the RDN. The aim of the present study was to investigate the role of SK channels in mediating the release of 5-HT in the amygdala. Apamin, a polypeptiditic compound with SK2-SK3 channel selectivity, was used to block the channels. A dual probing methodology with Nafion coated carbon-fiber micro-electrode (Nafion-mCFE) was implemented to measure concomitantly the extracellular levels of 5-HT in the amygdala and the firing rate of 5-HT neurons in the RDN of anesthetized rats. Subcutaneous administration of apamin increased both the extracellular 5-HT levels in the amygdala and the firing rate of RDN neurons at doses as low as 12.5 microg. The recorded RDN neurons were of 5-HT phenotype, according to electrophysiologic signature and to the effects observed with peripheral administration of 8-hydroxy-2-(d-n-propyl-amino) tetralin (8-OH-DPAT) a 5-HT(1A) agonist known to selectively reduce the firing of 5-HT neurons in RDN. Increases of extracellular 5-HT levels in the amygdala were also seen when apamin was microinjected into the RDN, suggesting a role for 5-HT neurons of the RDN as target for subcutaneously administered apamin. The confirmation of the involvement of 5-HT neurons projecting from RDN to the amygdala in mediating the effects of apamin was obtained by micro-infusion of tetradotoxine into the bundle of 5-HT ascending fibers located in the region of the posterior amygdala. Attenuation of 5-HT release in the amygdala was observed in presence of increased firing of 5-HT neurons of the RDN. In conclusion, the dual CFE micro-sensor probing approach was used to show that apamin increases 5-HT release in the amygdala by increasing the firing rate of 5-HT neurons in RDN.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

PubMed

Dinicola, Simona; Mariggiò, Maria Addolorata; Morabito, Caterina; Guarnieri, Simone; Cucina, Alessandra; Pasqualato, Alessia; D'Anselmi, Fabrizio; Proietti, Sara; Coluccia, Pierpaolo; Bizzarri, Mariano

2013-09-14

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Alteration of glutamate/GABA balance during acute alcohol intoxication in rats: effect of Xingnaojing injection.

PubMed

Wei, Jingjing; Yao, Limei; Yang, Lei; Zhao, Wei; Shi, Si; Cai, Qingyan; Chen, Dingsheng; Li, Weirong; Wang, Qi

2015-05-26

Xingnaojing Injection (XNJI) is a modern Chinese formula came from famous Chinese medicine An Gong Niu Huang Pill. XNJI has been used for treatment of cerebral diseases and stroke in China, and is approved by the State Food and Drug Administration of China for the treatment of acute alcohol intoxication (AAI). XNJI belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanisms of the XNJI effect on AAI. To investigate the effects of XNJI on glutamate, gamma-aminobutyric acid (GABA) and related receptor in lateral hypothalamic area (LHA) of AAI rat. Adult male Sprague-Dawley rats were implanted with microdialysis probes in LHA. Rats were randomly divided into control, model, 1.36mg/kg XNJI, 0.68mg/kg XNJI and 0.34mg/kg XNJI groups. During microdialysis, baseline samples were collected from 1h to 2.5h; thereafter, the rats were given an intraperitoneal injection of 52% ethanol, 5.2g/kg, or saline for control group. Twenty minutes later, three doses of XNJI was given by unilateral injection respectively, while saline for control and model groups, and samples were collected for the next 4h. The extracellular glutamate and GABA levels were measured in the LHA by a high performance liquid chromatography coupled with fluorescence detector (HPLC-FLU). The expression levels of related receptors N-methyl-d-aspartate receptor (NR) subunit NR2A, NR2B and GABAA were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Ethanol (5.2g/kg) significantly decreased the extracellular levels of glutamate and increased extracellular GABA in LHA. On the other hand ethanol significantly decreased NR2A and NR2B mRNAs expression, and increase GABAA mRNA expression. XNJI could increase the extracellular level of glutamate and decrease that of GABA; moreover, induced an increase in NR2A and NR2B mRNA expression, and a decrease in GABAA mRNA expression in LHA. The current changes in glutamate, GABA and mRNA expressions of related receptors in LHA after injection of XNJI suggest that changes in these neurotransmitters and receptors as a potential mechanism of action for AAI. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Critical role for the NLRP3 inflammasome during acute lung injury1

PubMed Central

Grailer, Jamison J.; Canning, Bethany A.; Kalbitz, Miriam; Haggadone, Mikel D.; Dhond, Rasika M.; Andjelkovic, Anuska V.; Zetoune, Firas S.; Ward, Peter A.

2014-01-01

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1β and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1β, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Antibody-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K+, increased [Ca+2]i, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1β levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug. PMID:24795455

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation

PubMed Central

Alberro, Ainhoa; Sáenz-Cuesta, Matías; Muñoz-Culla, Maider; Mateo-Abad, Maider; Gonzalez, Esperanza; Carrasco-Garcia, Estefania; Araúzo-Bravo, Marcos J.; Matheu, Ander; Vergara, Itziar; Otaegui, David

2016-01-01

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed “inflammaging” has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process. PMID:27447627

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation.

PubMed

Alberro, Ainhoa; Sáenz-Cuesta, Matías; Muñoz-Culla, Maider; Mateo-Abad, Maider; Gonzalez, Esperanza; Carrasco-Garcia, Estefania; Araúzo-Bravo, Marcos J; Matheu, Ander; Vergara, Itziar; Otaegui, David

2016-07-20

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

PubMed Central

Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

2016-01-01

Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

Zinc signaling in the hippocampus and its relation to pathogenesis of depression.

PubMed

Takeda, Atsushi

2012-06-01

Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons, a subclass of glutamatergic neurons. Zn(2+) serves as a signal factor in both the extracellular and intracellular compartments. Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of hippocampal function, i.e., cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation of CA1 LTP after exposure to acute stress. We propose that corticosterone-mediated increase in postsynaptic Zn(2+) signal, which is induced by acute stress, changes hippocampal function and then is possibly a risk factor under chronic stress circumstances to induce depressive symptoms. Copyright © 2012 Elsevier GmbH. All rights reserved.

Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation.

PubMed

Matsuo, Y; Kihara, T; Ikeda, M; Ninomiya, M; Onodera, H; Kogure, K

1995-11-01

A growing body of experimental data indicate that oxygen radicals may mediate the brain injury during ischemia-reperfusion. One potential source of oxygen radicals is activated neutrophils. To study the role of neutrophils in radical production during cerebral ischemia-reperfusion, we evaluated the effects of depletion of circulating neutrophils by administration of an anti-neutrophil monoclonal antibody (RP3) on radical formation in rats with 1-h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR decreased during MCA occlusion. After reperfusion, AR significantly increased at 30 min and 1 h, returned to near basal level until 2 h, and increased again at 24 h after reperfusion. In the rats treated with RP3, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, RP3 treatment completely inhibited the increase in extracellular AR after reperfusion. RP3 treatment exerted no effect on the changes in extracellular ascorbate or tissue PO2 throughout the experimental period. In conclusion, neutrophils are a major source of oxygen radicals during reperfusion after focal cerebral ischemia.

Evidence for involvement of nitric oxide and GABAB receptors in MK-801- stimulated release of glutamate in rat prefrontal cortex

PubMed Central

Roenker, Nicole L.; Gudelsky, Gary A.; Ahlbrand, Rebecca; Horn, Paul S.; Richtand, Neil M.

2012-01-01

Systemic administration of NMDA receptor antagonists elevates extracellular glutamate within prefrontal cortex. The cognitive and behavioral effects of NMDA receptor blockade have direct relevance to symptoms of schizophrenia, and recent studies demonstrate an important role for nitric oxide and GABAB receptors in mediating the effects of NMDA receptor blockade on these behaviors. We sought to extend those observations by directly measuring the effects of nitric oxide and GABAB receptor mechanisms on MK-801-induced glutamate release in the prefrontal cortex. Systemic MK-801 injection (0.3 mg/kg) to male Sprague-Dawley rats significantly increased extracellular glutamate levels in prefrontal cortex, as determined by microdialysis. This effect was blocked by pretreatment with the nitric oxide synthase inhibitor L-NAME (60 mg/kg). Reverse dialysis of the nitric oxide donor SNAP (0.5 – 5 mM) directly into prefrontal cortex mimicked the effect of systemic MK-801, dose-dependently elevating cortical extracellular glutamate. The effect of MK-801 was also blocked by systemic treatment with the GABAB receptor agonist baclofen (5 mg/kg). In combination, these data suggest increased nitric oxide formation is necessary for NMDA antagonist-induced elevations of extracellular glutamate in the prefrontal cortex. Additionally, the data suggest GABAB receptor activation can modulate the NMDA antagonist-induced increase in cortical glutamate release. PMID:22579658

Stimulation of inorganic pyrophosphate elaboration by cultured cartilage and chondrocytes.

PubMed

Ryan, L M; Kurup, I; Rosenthal, A K; McCarty, D J

1989-08-01

Inorganic pyrophosphate elaboration by articular cartilage may favor calcium pyrophosphate dihydrate crystal deposition. Frequently crystal deposits form in persons affected with metabolic diseases. The cartilage organ culture system was used to model these metabolic conditions while measuring the influence on extracellular pyrophosphate elaboration. Alterations of ambient pH, thyroid stimulating hormone levels, and parathyroid hormone levels did not change pyrophosphate accumulation in the media. However, subphysiologic ambient calcium concentrations (25, 100, 500 microM) increased pyrophosphate accumulation about chondrocytes 3- to 10-fold. Low calcium also induced release of [14C]adenine-labeled nucleotides from chondrocytes, potential substrates for generation of extracellular pyrophosphate by ectoenzymes. Exposing cartilage to 10% fetal bovine serum also enhanced by 50% the egress of inorganic pyrophosphate from the tissue.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

PubMed Central

Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

2014-01-01

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

PubMed Central

Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

2012-01-01

BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

NASA Astrophysics Data System (ADS)

Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

2017-08-01

Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

3,4-methylenedioxymethamphetamine self-administration is abolished in serotonin transporter knockout mice.

PubMed

Trigo, José Manuel; Renoir, Thibault; Lanfumey, Laurence; Hamon, Michel; Lesch, Klaus-Peter; Robledo, Patricia; Maldonado, Rafael

2007-09-15

The neurobiological mechanism underlying the reinforcing effects of 3,4-methylenedioxymethamphetamine (MDMA) remains unclear. The aim of the present study was to determine the contribution of the serotonin transporter (SERT) in MDMA self-administration behavior by using knockout (KO) mice deficient in SERT. Knockout mice and wild-type (WT) littermates were trained to acquire intravenous self-administration of MDMA (0, .03, .06, .125, and .25 mg/kg/infusion) on a fixed ratio 1 (FR1) schedule of reinforcement. Additional groups of mice were trained to obtain food and water to rule out operant responding impairments. Microdialysis studies were performed to evaluate dopamine (DA) and serotonin (5-HT) extracellular levels in the nucleus accumbens (NAC) and prefrontal cortex (PFC), respectively, after acute MDMA (10 mg/kg). None of the MDMA doses tested maintained intravenous self-administration in KO animals, whereas WT mice acquired responding for MDMA. Acquisition of operant responding for food and water was delayed in KO mice, but no differences between genotypes were observed on the last day of training. MDMA increased DA extracellular levels to a similar extent in the NAC of WT and KO mice. Conversely, extracellular concentrations of 5-HT in the PFC were increased following MDMA only in WT mice. These findings provide evidence for the specific involvement of SERT in MDMA reinforcing properties.

Changes in extracellular muscle volume affect heart rate and blood pressure responses to static exercise

NASA Astrophysics Data System (ADS)

Baum, K.; Essfeld, D.; Stegemann, J.

To investigate the effect of μg-induced peripheral extracellular fluid reductions on heart rate and blood pressure during isometric exercise, six healthy male subjects performed three calf ergometer test with different extracellular volumes of working muscles. In all tests, body positions during exercise were identical (supine with the knee joint flexed to 900). After a pre-exercise period of 25 min, during which calf volumes were manipulated, subjects had to counteract an external force of 180 N for 5 min. During the pre-exercise period three different protocols were applied. Test A: Subjects rested in the exercise position; test B: Body position was the same as in A but calf volume was increased by venous congestion (cuffs inflated to 80 mm Hg); test C: Calf volumes were decreased by a negative hydrostatic pressure (calves about 40 cm above heart level with the subjects supine). To clamp the changed calf volumes in tests B and C, cuffs were inflated to 300 mm Hg 5 min before the onset of exercise. This occlusion was maintained until termination of exercise. Compared to tests A and B, the reduced volume of test C led to significant increases in heart rate and blood pressure during exercise. Oxygen uptake did not exceed resting levels in B and C until cuffs were deflated, indicating that exclusively calf muscles contributed to the neurogenic peripheral drive. It is concluded that changes in extracellular muscle volume have to be taken into account when comparing heart rate and blood pressure during lg- and μg- exercise.

Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients.

PubMed

Logozzi, Mariantonia; Angelini, Daniela F; Iessi, Elisabetta; Mizzoni, Davide; Di Raimo, Rossella; Federici, Cristina; Lugini, Luana; Borsellino, Giovanna; Gentilucci, Alessandro; Pierella, Federico; Marzio, Vittorio; Sciarra, Alessandro; Battistini, Luca; Fais, Stefano

2017-09-10

Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Glutamine Acts as a Neuroprotectant against DNA Damage, Beta-Amyloid and H2O2-Induced Stress

PubMed Central

Chen, Jianmin; Herrup, Karl

2012-01-01

Glutamine is the most abundant free amino acid in the human blood stream and is ‘conditionally essential’ to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS). Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H2O2, zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD) brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD. PMID:22413000

Ethanol drinking reduces extracellular dopamine levels in the posterior ventral tegmental area of nondependent alcohol-preferring rats.

PubMed

Engleman, Eric A; Keen, Elizabeth J; Tilford, Sydney S; Thielen, Richard J; Morzorati, Sandra L

2011-09-01

Moderate ethanol exposure produces neuroadaptive changes in the mesocorticolimbic dopamine (DA) system in nondependent rats and increases measures of DA neuronal activity in vitro and in vivo. Moreover, moderate ethanol drinking and moderate systemic exposure elevates extracellular DA levels in mesocorticolimbic projection regions. However, the neuroadaptive changes subsequent to moderate ethanol drinking on basal DA levels have not been investigated in the ventral tegmental area (VTA). In the present study, adult female alcohol-preferring (P) rats were divided into alcohol-naive, alcohol-drinking, and alcohol-deprived groups. The alcohol-drinking group had continuous access to water and ethanol (15%, vol/vol) for 8 weeks. The alcohol-deprived group had 6 weeks of access followed by 2 weeks of ethanol deprivation, 2 weeks of ethanol re-exposure, followed again by 2 weeks of deprivation. The deprived rats demonstrated a robust alcohol deprivation effect (ADE) on ethanol reinstatement. The alcohol-naïve group had continuous access to water only. In the last week of the drinking protocol, all rats were implanted with unilateral microdialysis probes aimed at the posterior VTA and no-net-flux microdialysis was conducted to quantify extracellular DA levels and DA clearance. Results yielded significantly lower basal extracellular DA concentrations in the posterior VTA of the alcohol-drinking group compared with the alcohol-naive and alcohol-deprived groups (3.8±0.3nM vs. 5.0±0.5nM [P<.02] and 4.8±0.4nM, [P<.05], respectively). Extraction fractions were significantly (P<.0002) different between the alcohol-drinking and alcohol-naive groups (72±2% vs. 46±4%, respectively) and not significantly different (P=.051) between alcohol-deprived and alcohol-naive groups (61±6% for the alcohol-deprived group). The data indicate that reductions in basal DA levels within the posterior VTA occur after moderate chronic ethanol intake in nondependent P rats. This reduction may result, in part, from increased DA uptake and may be important for the maintenance of ethanol drinking. These adaptations normalize with ethanol deprivation and may not contribute to the ADE. Copyright © 2011 Elsevier Inc. All rights reserved.

Role of copper transport protein Antioxidant-1 in Angiotensin II-induced hypertension: A key regulator of Extracellular SOD

PubMed Central

Ozumi, Kiyoshi; Sudhahar, Varadarajan; Kim, Ha Won; Chen, Gin-Fu; Kohno, Takashi; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D.; Ushio-Fukai, Masuko; Fukai, Tohru

2012-01-01

Extracellular superoxide dismutase (SOD3) is a secretory copper enzyme involved in protecting angiotensin II (Ang II)-induced hypertension. We previously found that Ang II upregulates SOD3 expression and activity as a counter-regulatory mechanism; however, underlying mechanisms are unclear. Antioxidant-1 (Atox1) is shown to act as a copper-dependent transcription factor as well as copper chaperone for SOD3 in vitro, but its role in Ang II-induced hypertension in vivo is unknown. Here we show that Ang II infusion increases Atox1 expression as well as SOD3 expression and activity in aortas of wild-type mice, which are inhibited in mice lacking Atox1. Accordingly, Ang II increases vascular O2•− production, reduces endothelium-dependent vasodilation and increases vasoconstriction in mesenteric arterioles to a greater extent in Atox1−/− than in wild-type mice. This contributes to augmented hypertensive response to Ang II in Atox1−/− mice. In cultured vascular smooth muscle cells, Ang II promotes translocation of Atox1 to the nucleus, thereby increasing SOD3 transcription by binding to Atox1 responsive element in the SOD3 promoter. Furthermore, Ang II increases Atox1 binding to the copper exporter ATP7A which obtains copper from Atox1 as well as translocation of ATP7A to plasma membranes where it colocalizes with SOD3. As its consequence, Ang II decreases vascular copper levels, which is inhibited in Atox1−/− mice. In summary, Atox1 functions to prevent Ang II-induced endothelial dysfunction and hyper-contraction in resistant vessels as well as hypertension in vivo by reducing extracellular O2•− levels via increasing vascular SOD3 expression and activity. PMID:22753205

Developmental trajectory of contextual learning and 24-h acetylcholine release in the hippocampus

PubMed Central

Takase, Kenkichi; Sakimoto, Yuya; Kimura, Fukuko; Mitsushima, Dai

2014-01-01

To determine the developmental trajectory of hippocampal function in rats, we examined 24-h changes in extracellular acetylcholine (ACh) levels and contextual learning performance. Extracellular ACh significantly correlated with spontaneous behavior, exhibiting a 24-h rhythm in juvenile (4-week-old), pubertal (6-week-old), and adult (9- to 12-week-old) rats. Although juveniles of both sexes exhibited low ACh levels, adult males had higher ACh levels than adult females. Moreover, juveniles exhibited much more spontaneous activity than adults when they showed equivalent ACh levels. Similarly, juveniles of both sexes exhibited relatively low contextual learning performance. Because contextual learning performance was significantly increased only in males, adult males exhibited better performance than adult females. We also observed a developmental relationship between contextual learning and ACh levels. Scopolamine pretreatment blocked contextual learning and interrupted the correlation. Since long-term scopolamine treatment after weaning impaired contextual learning in juveniles, the cholinergic input may participate in the development of hippocampus. PMID:24435246

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

PubMed

Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

2017-09-25

Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Agmatine Prevents Adaptation of the Hippocampal Glutamate System in Chronic Morphine-Treated Rats.

PubMed

Wang, Xiao-Fei; Zhao, Tai-Yun; Su, Rui-Bin; Wu, Ning; Li, Jin

2016-12-01

Chronic exposure to opioids induces adaptation of glutamate neurotransmission, which plays a crucial role in addiction. Our previous studies revealed that agmatine attenuates opioid addiction and prevents the adaptation of glutamate neurotransmission in the nucleus accumbens of chronic morphine-treated rats. The hippocampus is important for drug addiction; however, whether adaptation of glutamate neurotransmission is modulated by agmatine in the hippocampus remains unknown. Here, we found that continuous pretreatment of rats with ascending doses of morphine for 5 days resulted in an increase in the hippocampal extracellular glutamate level induced by naloxone (2 mg/kg, i.p.) precipitation. Agmatine (20 mg/kg, s.c.) administered concurrently with morphine for 5 days attenuated the elevation of extracellular glutamate levels induced by naloxone precipitation. Furthermore, in the hippocampal synaptosome model, agmatine decreased the release and increased the uptake of glutamate in synaptosomes from chronic morphine-treated rats, which might contribute to the reduced elevation of glutamate levels induced by agmatine. We also found that expression of the hippocampal NR2B subunit, rather than the NR1 subunit, of N-methyl-D-aspartate receptors (NMDARs) was down-regulated after chronic morphine treatment, and agmatine inhibited this reduction. Taken together, agmatine prevented the adaptation of the hippocampal glutamate system caused by chronic exposure to morphine, including modulating extracellular glutamate concentration and NMDAR expression, which might be one of the mechanisms underlying the attenuation of opioid addiction by agmatine.

Effect of O-methyl-β-cyclodextrin-modified magnetic nanoparticles on the uptake and extracellular level of l-glutamate in brain nerve terminals.

PubMed

Horák, Daniel; Beneš, Milan; Procházková, Zuzana; Trchová, Miroslava; Borysov, Arsenii; Pastukhov, Artem; Paliienko, Konstantin; Borisova, Tatiana

2017-01-01

Changes in cholesterol concentration in the plasma membrane of presynaptic nerve terminals nonspecifically modulate glutamate transport and homeostasis in the central nervous system. Reduction of the cholesterol content in isolated rat brain nerve terminals (synaptosomes) using cholesterol-depleting agents decreases the glutamate uptake and increases the extracellular level of glutamate in nerve terminals. Extraction of cholesterol from the plasma membrane and its further removal from the synaptosomes by external magnetic field can be achieved by means of magnetic nanoparticles with immobilized cholesterol-depleting agent such as O-methyl-β-cyclodextrin (MCD). A simple approach is developed for preparation of maghemite (γ-Fe 2 O 3 ) nanoparticles containing chemically bonded MCD. The method is based on preparation of a silanization agent containing MCD. It is synthesized by the reaction of triethoxy(3-isocyanatopropyl)silane with MCD. Base-catalyzed silanization of superparamagnetic γ-Fe 2 O 3 provides a relatively stable colloid product containing 48μmol of MCDg -1 . MCD-modified γ-Fe 2 O 3 nanoparticles decrease the initial rate of the uptake and accumulation of l-[ 14 C]glutamate and increase the extracellular l-[ 14 C]glutamate level in the preparation of nerve terminals. The effect of MCD-immobilized nanoparticles is the same as that of MCD solution; moreover, magnetic manipulation of the nanoparticles enables removal of bonded cholesterol. Copyright © 2016 Elsevier B.V. All rights reserved.

Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection

PubMed Central

Break, Timothy J.; Jun, Sujung; Indramohan, Mohanalaxmi; Carr, Karen D.; Sieve, Amy N.; Dory, Ladislav; Berg, Rance E.

2012-01-01

Reactive oxygen and nitrogen species (ROS/RNS) play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of ROS/RNS and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the present study, we utilized mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that, despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively impacted host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased co-localization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent nitric oxide (NO·) when compared to mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO3·−) in livers from mice lacking ecSOD compared to mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared to neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention. PMID:22393157

Faster flux of neurotransmitter glutamate during seizure — Evidence from 13C-enrichment of extracellular glutamate in kainate rat model

PubMed Central

2017-01-01

The objective is to examine how the flux of neurotransmitter glutamate from neurons to the extracellular fluid, as measured by the rate of 13C enrichment of extracellular glutamate (GLUECF), changes in response to seizures in the kainate-induced rat model of temporal-lobe epilepsy. Following unilateral intrahippocampal injection of kainate, GLUECF was collected by microdialysis from the CA1/CA3 region of awake rats, in combination with EEG recording of chronic-phase recurrent seizures and intravenous infusion of [2,5-13C]glucose. The 13C enrichment of GLUECF C5 at ~ 10 picomol level was measured by gas-chromatography mass-spectrometry. The rate of 13C enrichment, expressed as the increase of the fractional enrichment/min, was 0.0029 ± 0.0001/min in frequently seizing rats (n = 4); this was significantly higher (p < 0.01) than in the control (0.00167 ± 0.0001/min; n = 6) or in rats with infrequent seizures (0.00172 ± 0.0001/min; n = 6). This result strongly suggests that the flux of the excitatory neurotransmitter from neurons to the extracellular fluid is significantly increased by frequent seizures. The extracellular [12C + 13C]glutamate concentration increased progressively in frequently seizing rats. Taken together, these results strongly suggest that the observed seizure-induced high flux of glutamate overstimulated glutamate receptors, which triggered a chain reaction of excitation in the CA3 recurrent glutamatergic networks. The rate of 13C enrichment of extracellular glutamine (GLNECF) at C5 was 0.00299 ± 0.00027/min in frequently seizing rats, which was higher (p < 0.05) than in controls (0.00227 ± 0.00008/min). For the first time in vivo, this study examined the effects of epileptic seizures on fluxes of the neurotransmitter glutamate and its precursor glutamine in the extracellular fluid of the hippocampus. The advantages, limitations and the potential for improvement of this approach for pre-clinical and clinical studies of temporal-lobe epilepsy are discussed. PMID:28403176

Estrous cycle modulation of extracellular serotonin in mediobasal hypothalamus: role of the serotonin transporter and terminal autoreceptors.

PubMed

Maswood, S; Truitt, W; Hotema, M; Caldarola-Pastuszka, M; Uphouse, L

1999-06-12

In vivo microdialysis was used to examine extracellular serotonin (5-HT) in the mediobasal hypothalamus (MBH) of male and female Fischer (CDF-344) rats. Females from the stages of diestrus, proestrus, and estrus were used. Additionally, ovariectomized rats, primed subcutaneously (s.c.) with estradiol benzoate or estradiol benzoate plus progesterone were examined. Extracellular 5-HT in the MBH varied with stage of the estrous cycle and with the light/dark cycle. Proestrous females had the highest microdialysate concentrations of 5-HT during the light portion of the light/dark cycle and lowest concentrations during the dark portion of the cycle. Diestrous females had the highest levels during the dark portion of the cycle, while males and estrous females showed little change between light and dark portions of the cycle. In ovariectomized rats, there was no effect of 2.5 microg or 25 microg estradiol benzoate (s.c.) on extracellular 5-HT; but the addition of 500 microg progesterone, 48 h after estrogen priming, reduced microdialysate 5-HT near the threshold for detection. In intact females and in males, reverse perfusion with 3 microM fluoxetine, a selective serotonin reuptake inhibitor (SSRI), or 2 microM methiothepin, a 5-HT receptor antagonist, increased microdialysate concentrations of 5-HT. Estrous females and males showed nearly a 4-fold increase in microdialysate 5-HT in response to fluoxetine while smaller responses were seen in diestrous and proestrous rats. In contrast, proestrous rats showed the largest response to methiothepin. Estrous females showed a delayed response to methiothepin, but there was no methiothepin-induced increase in extracellular 5-HT in males. These findings are discussed in reference to the suggestion that extracellular 5-HT in the MBH is regulated in a manner that is gender and estrous cycle dependent. The 5-HT terminal autoreceptor may exert a greater role in proestrous females; the serotonin transporter appears to play a more active role in the regulation of extracellular 5-HT in estrous females and in males. Copyright 1999 Elsevier Science B.V.

Physiological Role of Gap-Junctional Hemichannels

PubMed Central

Quist, Arjan Pieter; Rhee, Seung Keun; Lin, Hai; Lal, Ratneshwar

2000-01-01

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation. PMID:10704454

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones.

PubMed

Mark, Christina; Zór, Kinga; Heiskanen, Arto; Dufva, Martin; Emnéus, Jenny; Finnie, Christine

2016-12-15

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts. Copyright © 2016 Elsevier Inc. All rights reserved.

Recombinant HE4 protein promotes proliferation of pancreatic and endometrial cancer cell lines.

PubMed

Lu, Qinsheng; Chen, Haibin; Senkowski, Christopher; Wang, Jianhao; Wang, Xue; Brower, Steven; Glasgow, Wayne; Byck, David; Jiang, Shi-Wen; Li, Jinping

2016-01-01

Pancreatic adenocarcinoma is one of the most deadly malignancies, and endometrial cancer represents the most common gynecologic cancer in the USA. Better understanding on the pathologic mechanisms and pathways is required for effective treatment of these malignancies. Recently, human epididymis protein 4 (HE4 or WFDC2), a secretory glycoprotein, was found to be overexpressed in pancreatic and endometrial cancers. In addition, studies have shown that HE4 overexpression in endometrial cancer cell lines led to faster cancer progression in a mouse subcutaneous model. These findings raise a question on the role(s) of secretory, extracellular HE4 in cancer development. In the present study, we found that treatment of pancreatic and endometrial cancer cell lines with purified, extracellular HE4 protein led to a significant increase in cell viability and proliferation. Moreover, extracellular HE4 protein was able to increase DNA synthesis, and modulate the mRNA and protein levels of cell cycle marker PCNA and cell cycle inhibitor p21. These effects appeared to be robust and sustainable and required a relatively low concentration of HE4 protein. The findings indicated the secreted, extracellular HE4 may carry some physiopathological functions. Via paracrine/endocrine actions, circulatory HE4 produced by malignant cells may contribute to pancreatic and endometrial cancer progression and/or metastasis.

Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain

NASA Technical Reports Server (NTRS)

Owan, I.; Burr, D. B.; Turner, C. H.; Qiu, J.; Tu, Y.; Onyia, J. E.; Duncan, R. L.

1997-01-01

Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.

High capacity for extracellular acid-base regulation in the air-breathing fish Pangasianodon hypophthalmus.

PubMed

Damsgaard, Christian; Gam, Le Thi Hong; Tuong, Dang Diem; Thinh, Phan Vinh; Huong Thanh, Do Thi; Wang, Tobias; Bayley, Mark

2015-05-01

The evolution of accessory air-breathing structures is typically associated with reduction of the gills, although branchial ion transport remains pivotal for acid-base and ion regulation. Therefore, air-breathing fishes are believed to have a low capacity for extracellular pH regulation during a respiratory acidosis. In the present study, we investigated acid-base regulation during hypercapnia in the air-breathing fish Pangasianodon hypophthalmus in normoxic and hypoxic water at 28-30°C. Contrary to previous studies, we show that this air-breathing fish has a pronounced ability to regulate extracellular pH (pHe) during hypercapnia, with complete metabolic compensation of pHe within 72 h of exposure to hypoxic hypercapnia with CO2 levels above 34 mmHg. The high capacity for pHe regulation relies on a pronounced ability to increase levels of HCO3(-) in the plasma. Our study illustrates the diversity in the physiology of air-breathing fishes, such that generalizations across phylogenies may be difficult. © 2015. Published by The Company of Biologists Ltd.

Early metabolic responses to lithium/pilocarpine-induced status epilepticus in rat brain.

PubMed

Imran, Imran; Hillert, Markus H; Klein, Jochen

2015-12-01

The lithium-pilocarpine model of status epilepticus is a well-known animal model of temporal lobe epilepsy. We combined this model with in vivo microdialysis to investigate energy metabolites and acute cellular membrane damage during seizure development. Rats were implanted with dialysis probes and pretreated with lithium chloride (127 mg/kg i.p.). Twenty-four hours later, they received pilocarpine (30 mg/kg s.c.) which initiated seizures within 30 min. In the dialysate from rat hippocampus, we observed a transient increase in glucose and a prominent, five-fold increase in lactate during seizures. Lactate release was because of neuronal activation as it was strongly reduced by infusion of tetrodotoxin, administration of atropine or when seizures were terminated by diazepam or ketamine. In ex vivo assays, mitochondrial function as measured by respirometry was not affected by 90 min of seizures. Extracellular levels of choline, however, increased two-fold and glycerol levels 10-fold, which indicate cellular phospholipid breakdown during seizures. Within 60 min of pilocarpine administration, hydroxylation of salicylate increased two-fold and formation of isoprostanes 20-fold, revealing significant oxidative stress in hippocampal tissue. Increases in lactate, glycerol and isoprostanes were abrogated, and increases in choline were completely prevented, when hippocampal probes were perfused with calcium-free solution. Similarly, administration of pregabalin (100 mg/kg i.p.), a calcium channel ligand, 15 min prior to pilocarpine strongly attenuated parameters of membrane damage and oxidative stress. We conclude that seizure development in a rat model of status epilepticus is accompanied by increases in extracellular lactate, choline and glycerol, and by oxidative stress, while mitochondrial function remains intact for at least 90 min. Membrane damage depends on calcium influx and can be prevented by treatment with pregabalin. Status epilepticus (SE) was induced in rats by lithium-pilocarpine ('Pilo') administration, and extracellular metabolites were measured by microdialysis. Seizures caused several-fold increases in lactate levels which were attenuated by diazepam ('Diaz'), ketamine, atropine and tetrodotoxin (TTX). Indicators of oxidative stress and membrane damage were also increased during seizures. Omission of calcium and pregabalin, a calcium channel blocker, reduced cellular damage induced by SE. © 2015 International Society for Neurochemistry.

Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

PubMed Central

Keibler, Mark A.; Park, Donglim Esther; Molla, Vadim; Cheng, Jingwei; Stephanopoulos, Gregory

2016-01-01

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis. PMID:27880818

Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro.

PubMed

Miyanishi, Keita; Trindade, Michael C D; Lindsey, Derek P; Beaupré, Gary S; Carter, Dennis R; Goodman, Stuart B; Schurman, David J; Smith, R Lane

2006-06-01

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Monoaminergic Psychomotor Stimulants: Discriminative Stimulus Effects and Dopamine Efflux

PubMed Central

Desai, Rajeev I.; Paronis, Carol A.; Martin, Jared; Desai, Ramya

2010-01-01

The present studies were conducted to investigate the relationship between discriminative stimulus effects of indirectly acting monoaminergic psychostimulants and their ability to increase extracellular levels of dopamine (DA) in the nucleus accumbens (NAcb) shell. First, the behavioral effects of methamphetamine (MA), cocaine (COC), 1-[2-[bis(4-fluorophenyl-)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909), d-amphetamine, and methylphenidate were established in rats trained to discriminate intraperitoneal injections of 0.3 mg/kg MA from saline. In other studies, in vivo microdialysis was used to determine the effects of MA, COC, and GBR 12909 on extracellular DA levels in the NAcb shell. Results show that all drugs produced dose-related and full substitution for the discriminative stimulus effects of 0.3 mg/kg MA. In microdialysis studies, cumulatively administered MA (0.3–3 mg/kg), COC (3–56 mg/kg), and GBR 12909 (3–30 mg/kg) produced dose-dependent increases in DA efflux in the NAcb shell to maxima of approximately 1200 to 1300% of control values. The increase in DA levels produced by MA and COC was rapid and short-lived, whereas the effect of GBR 12909 was slower and longer lasting. Dose-related increases in MA lever selection produced by MA, COC, and GBR 12909 corresponded with graded increases in DA levels in the NAcb shell. Doses of MA, COC, and GBR 12909 that produced full substitution increased DA levels to approximately 200 to 400% of control values. Finally, cumulatively administered MA produced comparable changes in DA levels in both naive and 0.3 mg/kg MA-trained rats. These latter results suggest that sensitization of DA release does not play a prominent role in the discriminative stimulus effects of psychomotor stimulants. PMID:20190012

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stuart, Rhona K.; Mayali, Xavier; Boaro, Amy A.

2016-06-28

Although it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of 13C-labeled and 15N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively,more » of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM. IMPORTANCECyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.« less

Impacts of Rac- and S-metolachlor on cyanobacterial cell integrity and release of microcystins at different nitrogen levels.

PubMed

Wang, Jia; Zhang, Lijuan; Fan, Jiajia; Wen, Yuezhong

2017-08-01

Pesticide residues and nitrogen overload (which caused cyanobacteria blooms) have been two serious environmental concerns. In particular, chiral pesticides with different structures may have various impacts on cyanobacteria. Nitrogen may affect the behavior between pesticides and cyanobacteria (e.g., increase the adverse effects of pesticides on cyanobacteria). This study evaluated the impacts of Rac- and S-metolachlor on the cell integrity and toxin release of Microcystis aeruginosa cells at different nitrogen levels. The results showed that (both of the configurations: Rac-, S-) metolachlor could inhibit M. aeruginosa cell growth under most conditions, and the inhibition rates were increased with the growing concentrations of nitrogen and metolachlor. However, cyanobacterial growth was promoted in 48 h under environmental relevant condition (1 mg/L metolachlor and 0.15 mg/L nitrogen). Therefore, the water authorities should adjust the treatment parameters to remove possible larger numbers of cyaonbacteria under that condition. On the other hand, the inhibition degree of M. aeruginosa cell growth by S-metolachlor treatments was obviously larger than Rac-metolachlor treatments. S-metolachlor also had a stronger ability in compromising M. aeruginosa cells than Rac-metolachlor treatments. Compared to control samples, more extracellular toxins (12%-86% increases) were detected after 5 mg/L S-metolachlor treatment for 72 h at different nitrogen levels, but the variations of extracellular toxins caused by 5 mg/L Rac-metolachlor addition could be neglected. Consequently, higher concentrations of metolachlor in source waters are harmful to humans, but it may prevent cyanobacterial blooms. However, the potential risks (e.g. build-up of extracellular toxins) should be considered. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells.

PubMed

Fukushima, Yoshimi; Okamoto, Masaaki; Ishikawa, Kana; Kouwaki, Takahisa; Tsukamoto, Hirotake; Oshiumi, Hiroyuki

2018-06-07

Pattern-recognition receptors (PRRs) recognizes viral RNAs and trigger the innate immune responses. Toll-like receptor 3 (TLR3), a PRR, recognizes viral double-stranded RNA (dsRNA) in endolysosomes, whereas cytoplasmic dsRNA is sensed by another PRR, MDA5. TLR3 and MDA5 utilize TICAM-1 and MAVS, respectively, to trigger the signal for inducing innate immune responses. Extracellular vesicles (EVs) include the exosomes and microvesicles; an accumulating body of evidence has shown that EVs delivers functional RNA, such as microRNAs (miRNAs), to other cells and thus mediate intercellular communications. Therefore, EVs carrying miRNAs affect innate immune responses in macrophages and dendritic cells. However, the mechanism underlying the regulation of miRNA levels in EVs remains unclear. To elucidate the mechanism, we sought to reveal the pathway that control miRNA expression levels in EVs. Here, we found that TLR3 stimulation increased miR-21 levels in EVs released from various types of human cells. Ectopic expression of the TLR3 adaptor, TICAM-1, increased miR-21 levels in EVs but not intracellular miR-21 levels, suggesting that TICAM-1 augmented sorting of miR-21 to EVs. In contrast, the MDA5 adaptor, MAVS, did not increase miR-21 levels in EVs. The siRNA for TICAM-1 reduced EV miR-21 levels after stimulation of TLR3. Collectively, our data indicate a novel role of the TLR3-TICAM-1 pathway in controlling miR-21 levels in EVs. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline.

PubMed Central

Miyata, S; Monnier, V

1992-01-01

Pyrraline is one of the major Maillard compounds resulting from the reaction of glucose with amino compounds at slightly acidic pH. For in vivo studies, monoclonal pyrraline antibodies were raised after immunization of Balb/c mice with keyhole limpet hemocyamin-caproyl pyrraline conjugate. Of 660 hybridoma clones from one donor, 260 produced an antibody to the free hapten, two of which named Pyr-A and Pyr-B also cross-reacted with L-lysyl pyrraline. Using Pyr-B antibody and an ELISA, a gradual increase in pyrraline immunoreactivity was observed in serum albumin incubated with glucose or 3-deoxyglucosone. Plasma pyrraline levels increased fourfold (P less than 0.001) in Sprague-Dawley rats upon induction of diabetes with streptozotocin and were twofold increased in randomly selected plasmas from diabetic humans. Highly specific pyrraline immunoreactivity was detected in sclerosed glomeruli from diabetic and old normal kidneys as well as in renal arteries with arteriolosclerosis and in perivascular and peritubular sclerosed extracellular matrix and basement membranes. The preferential localization of pyrraline immunoreactivity in the extracellular matrix strengthens the notion that the advanced glycosylation reaction may contribute to decreased turnover and thickening of the extracellular matrix in diabetes and aging. Images PMID:1556177

Peroxidase Release Induced by Ozone in Sedum album Leaves

PubMed Central

Castillo, Federico J.; Penel, Claude; Greppin, Hubert

1984-01-01

The effect of ozone was studied on the peroxidase activity from various compartments of Sedum album leaves (epidermis, intercellular fluid, residual cell material, and total cell material). The greatest increase following a 2-hour ozone exposure (0.4 microliters O3 per liter) was observed in extracellular peroxidases. Most of the main bands of peroxidase activity separated by isoelectric focusing exhibited an increase upon exposure to ozone. Incubation experiments with isolated peeled or unpeeled leaves showed that leaves from ozone-treated plants release much more peroxidases in the medium than untreated leaves. The withdrawal of Ca2+ ions reduced the level of extracellular peroxidase activity either in whole plants or in incubation experiments. This reduction and the activation obtained after addition of Ca2+ resulted from a direct requirement of Ca2+ by the enzyme and from an effect of Ca2+ on peroxidase secretion. The ionophore A23187 promoted an increase of extracellular peroxidase activity only in untreated plants. The release of peroxidases by untreated and ozone-treated leaves is considerably lowered by metabolic inhibitors (3-(3,4-dichlorophenyl)-1,1-dimethylurea and sodium azide) and by puromycin. Images Fig. 1 PMID:16663520

Association of extracellular cleavage of E-cadherin mediated by MMP-7 with HGF-induced in vitro invasion in human stomach cancer cells.

PubMed

Lee, K H; Choi, E Y; Hyun, M S; Jang, B I; Kim, T N; Kim, S W; Song, S K; Kim, J H; Kim, J-R

2007-01-01

Proteolytic shedding of the ectodomain of a variety of transmembrane proteins, including cell-to-cell adhesion molecules, has been observed in solid cancers. We have investigated whether extracellular cleavage of E-cadherin mediated by matrix metalloproteinase-7 (MMP-7) is involved in hepatocyte growth factor (HGF) induced in vitro invasion in stomach cancer cells. The effects of HGF on the expression of E-cadherin/beta-catenin and MMP-7 at both the protein and mRNA levels were assessed in stomach cancer cells, NUGC-3 and MKN-28, and in cells in which the expression of MMP-7 was downregulated by transfection with a MMP-7 short hairpin RNA plasmid. Treatment with HGF increased the extracellular cleavage of E-cadherin and the release of MMP-7 and reduced the level of E-cadherin in a dose- and time-dependent manner. HGF treatment repressed the phosphorylation of beta-catenin in a Triton-soluble fraction, but enhanced this phosphorylation in a Triton-insoluble fraction. The association of E-cadherin with beta-catenin was decreased by HGF treatment in the Triton-soluble fraction. In addition, treatment of MMP-7 short hairpin RNA transfected NUGC-3 cells with HGF resulted in no extracellular cleavage of E-cadherin and also decreased the in vitro cell invasion. These results suggest that incubation with HGF mediated the release of MMP-7, resulting in extracellular cleavage of E-cadherin from stomach cancer cells. This might be a key mechanism in HGF-induced in vitro invasion and metastasis. Copyright 2007 S. Karger AG, Basel.

Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

PubMed

Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

2017-08-01

Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Modulation of extracellular ATP content of mast cells and DRG neurons by irradiation: studies on underlying mechanism of low-level-laser therapy.

PubMed

Wang, Lina; Hu, Lei; Grygorczyk, Ryszard; Shen, Xueyong; Schwarz, Wolfgang

2015-01-01

Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

Accumbal strychnine-sensitive glycine receptors: an access point for ethanol to the brain reward system.

PubMed

Molander, Anna; Söderpalm, Bo

2005-01-01

Ethanol (EtOH), like other drugs of abuse, increases extracellular dopamine (DA) levels in the nucleus accumbens (nAc) of the brain reward system, an effect that may be of importance for alcohol addiction. How this DA increase is produced is not fully understood, although previous studies from the present laboratories indicate that nicotinic acetylcholine receptors in the ventral tegmental area play an important role in mediating this effect. Furthermore, activation of these receptors may be secondary to some priming effect produced by EtOH in the nAc. We recently demonstrated that strychnine-sensitive glycine receptors (GlyRs) are present in the nAc and that they are involved in regulating extracellular DA levels. Here we examine the tentative role of these accumbal GlyRs in the above-mentioned priming mechanism of EtOH. In vivo microdialysis (coupled to high pressure liquid chromatography with electrochemical detection) and reversed microdialysis, in awake, freely moving adult male Wistar rats. Local perfusion of strychnine decreased accumbal DA levels per se and completely prevented the increase of accumbal DA levels after both local and systemic EtOH administration. Accumbal perfusion of the GlyR agonist glycine instead increased DA levels in a subpopulation of rats and prevented the EtOH-induced increase after local but not systemic EtOH in all animals. The present results suggest that GlyRs in the nAc might constitute targets for EtOH in its mesolimbic DA-activating effect. Gene polymorphism and drug developmental studies that focus on this receptor population and its relation to alcohol dependence are warranted.

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

PubMed

Kato, Kosuke; Zemskova, Marina A; Hanss, Alec D; Kim, Marianne M; Summer, Ross; Kim, Kwang Chul

2017-11-25

MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. Altogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung. Copyright © 2017. Published by Elsevier Inc.

Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

PubMed

Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

2016-05-01

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction. Copyright © 2016 Elsevier B.V. All rights reserved.

Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C.

PubMed Central

Racke, F K; Nemeth, E F

1993-01-01

1. The effects of protein kinase C (PKC) activators and inhibitors on the mechanisms regulating cytosolic Ca2+ homeostasis in dissociated bovine parathyroid cells loaded with fura-2 were examined. 2. Stepwise increases in the concentration of extracellular Ca2+ (from 0.5 to 2 or 3 mM) elicited transient followed by sustained increases in the concentration of intracellular free Ca2+ ([Ca2+]i). Cytosolic Ca2+ transients reflected the mobilization of intracellular Ca2+ and influx of extracellular Ca2+ whereas sustained increases in [Ca2+]i resulted from the influx of extracellular Ca2+. Brief (1-2 min) pretreatment with phorbol myristate acetate (PMA) shifted the concentration-response curve for extracellular Ca(2+)-induced cytosolic Ca2+ transients to the right without affecting the maximal response. Cytosolic Ca2+ transients elicited by extracellular Mg2+ were similarly affected by PMA. 3. These effects of PMA were mimicked by various other activators of PKC with the rank order of potency PMA > phorbol dibutyrate > bryostatin , > (-)indolactam V > mezerein. Isomers or analogues of these compounds that do not alter PKC activity (4 alpha-phorbols and (+)indolactam V) did not alter [Ca2+]i. 4. PKC activators depressed evoked increases in [Ca2+]i when influx of extracellular Ca2+ was blocked with Gd3+. Cytosolic Ca2+ transients elicited by extracellular Mg2+ in the absence of extracellular Ca2+ were similarly inhibited by PKC activators. Activation of PKC thus inhibits the mobilization of intracellular Ca2+ elicited by extracellular divalent cations. 5. Increases in the concentration of extracellular Ca2+ caused corresponding increases in the formation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3). Pretreatment with PMA shifted the concentration-response curve for extracellular Ca(2+)-induced [3H]InsP3 formation to the right without affecting the maximal response. 6. PKC activators also caused some depression of steady-state increases in [Ca2+]i elicited by extracellular Ca2+. In contrast, PMA did not affect increases in [Ca2+]i elicited by ionomycin or thapsigargin. 7. Ba2+ was used to monitor divalent cation influx. PMA decreased the rate of rise of the fluorescent signal elicited by extracellular Ba2+. 8. All these effects of PKC activators on [Ca2+]i were blocked or reversed by staurosporine at concentrations (30-100 nM) that inhibited PKC activity in parathyroid cells. Staurosporine alone potentiated cytosolic Ca2+ responses evoked by submaximal concentrations of extracellular divalent cations. 9. PKC thus depresses both the mobilization of intracellular Ca2+ and the influx of extracellular Ca2+ in parathyroid cells. The effects on [Ca2+]i provide evidence for a Ca2+ receptor on the surface of parathyroid cells that uses transmembrane signalling mechanisms common to some other Ca(2+)-mobilizing receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8254504

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Type of carbohydrate in feed affects the expression of small leucine-rich proteoglycans (SLRPs), glycosaminoglycans (GAGs) and interleukins in skeletal muscle of Atlantic cod (Gadus morhua L.).

PubMed

Tingbø, M G; Pedersen, M E; Grøndahl, F; Kolset, S O; Veiseth-Kent, E; Enersen, G; Hannesson, K O

2012-09-01

Aquaculture requires feed that ensures rapid growth and healthy fish. Higher inclusion of plant ingredients is desirable, as marine resources are limited. In this study we investigated the effects of higher starch inclusion in feed on muscular extracellular matrix and interleukin expression in farmed cod. Starch was replaced by complex fibers in the low-starch diet to keep total carbohydrate inclusion similar. Blood glucose and fructosamine levels were elevated in the high-starch group. The group fed a high-starch diet showed up-regulation on mRNA level of proteoglycans biglycan and decorin. ELISA confirmed the real-time PCR results on protein level for biglycan and also showed increase of lumican. For decorin the protein levels were decreased in the high-starch group, in contrast to real-time PCR results. Disaccharide analyses using HPLC showed reduction of glycosaminoglycans. Further, there was up-regulation of interleukin-1β and -10 on mRNA level in muscle. This study shows that the muscular extracellular matrix composition is affected by diet, and that a high-starch diet results in increased expression of pro-inflammatory genes similar to diabetes in humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

PubMed Central

Eberhart, Lauren J.; Ohlert, Janet M.; Knutson, Carolann M.; Plunkett, Mary H.

2015-01-01

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. PMID:25888177

Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs.

PubMed

Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R; McIntosh, J Michael; Brunzell, Darlene H; Cannon, Jason R; Drenan, Ryan M

2014-04-01

α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. © 2013 International Society for Neurochemistry.

Nandrolone-induced aggressive behavior is associated with alterations in extracellular glutamate homeostasis in mice.

PubMed

Kalinine, Eduardo; Zimmer, Eduardo Rigon; Zenki, Kamila Cagliari; Kalinine, Iouri; Kazlauckas, Vanessa; Haas, Clarissa Branco; Hansel, Gisele; Zimmer, Aline Rigon; Souza, Diogo Onofre; Müller, Alexandre Pastoris; Portela, Luis Valmor

2014-07-01

Nandrolone decanoate (ND), an anabolic androgenic steroid (AAS), induces an aggressive phenotype by mechanisms involving glutamate-induced N-methyl-d-aspartate receptor (NMDAr) hyperexcitability. The astrocytic glutamate transporters remove excessive glutamate surrounding the synapse. However, the impact of supraphysiological doses of ND on glutamate transporters activity remains elusive. We investigated whether ND-induced aggressive behavior is interconnected with GLT-1 activity, glutamate levels and abnormal NMDAr responses. Two-month-old untreated male mice (CF1, n=20) were tested for baseline aggressive behavior in the resident-intruder test. Another group of mice (n=188) was injected with ND (15mg/kg) or vehicle for 4, 11 and 19days (short-, mid- and long-term endpoints, respectively) and was evaluated in the resident-intruder test. Each endpoint was assessed for GLT-1 expression and glutamate uptake activity in the frontoparietal cortex and hippocampal tissues. Only the long-term ND endpoint significantly decreased the latency to first attack and increased the number of attacks, which was associated with decreased GLT-1 expression and glutamate uptake activity in both brain areas. These alterations may affect extracellular glutamate levels and receptor excitability. Resident males were assessed for hippocampal glutamate levels via microdialysis both prior to, and following, the introduction of intruders. Long-term ND mice displayed significant increases in the microdialysate glutamate levels only after exposure to intruders. A single intraperitoneal dose of the NMDAr antagonists, memantine or MK-801, shortly before the intruder test decreased aggressive behavior. In summary, long-term ND-induced aggressive behavior is associated with decreased extracellular glutamate clearance and NMDAr hyperexcitability, emphasizing the role of this receptor in mediating aggression mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Extracellular 2′,3′-cAMP-adenosine pathway in proximal tubular, thick ascending limb, and collecting duct epithelial cells

PubMed Central

Gillespie, Delbert G.

2013-01-01

In a previous study, we demonstrated that human proximal tubular epithelial cells obtained from a commercial source metabolized extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP and extracellular 2′-AMP and 3′-AMP to adenosine (the extracellular 2′,3′-cAMP-adenosine pathway; extracellular 2′,3′-cAMP → 2′-AMP + 3′-AMP → adenosine). The purpose of this study was to investigate the metabolism of extracellular 2′,3′-cAMP in proximal tubular vs. thick ascending limb vs. collecting duct epithelial cells freshly isolated from their corresponding nephron segments obtained from rat kidneys. In epithelial cells from all three nephron segments, 1) extracellular 2′,3′-cAMP was metabolized to 2′-AMP and 3′-AMP, with 2′-AMP > 3′-AMP, 2) the metabolism of extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP was not inhibited by either 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) or 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor), 3) extracellular 2′,3′-cAMP increased extracellular adenosine levels, 4) 3′-AMP and 2′-AMP were metabolized to adenosine with an efficiency similar to that of 5′-AMP, and 5) the metabolism of 5′-AMP, 3′-AMP, and 2′-AMP was not inhibited by α,β-methylene-adenosine-5′-diphosphate (CD73 inhibitor). These results support the conclusion that renal epithelial cells all along the nephron can metabolize extracellular 2′,3′-cAMP to 2′-AMP and 3′-AMP and can efficiently metabolize extracellular 2′-AMP and 3′-AMP to adenosine and that the metabolic enzymes involved are not the classical phosphodiesterases nor ecto-5′-nucleotidase (CD73). Because 2′,3′-cAMP is released by injury and because previous studies demonstrate that the extracellular 2′,3′-cAMP-adenosine pathway stimulates epithelial cell proliferation via adenosine A2B receptors, the present results suggest that the extracellular 2′,3′-cAMP-adenosine pathway may help restore epithelial cells along the nephron following kidney injury. PMID:23077101

Some medicinal plants as immunostimulant for fish.

PubMed

Dügenci, Süheyla Karataş; Arda, Nazli; Candan, Akin

2003-09-01

Immunostimulant effects of the dietary intake of various medicinal plant extracts on fish, rainbow trout (Oncorhynchus mykiss), were investigated. For this purpose fish were fed with diets containing aqueous extracts of mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Food containing lyophilized extracts of these plants as 0.1 and 1% was used at a rate of 2% of body weight per day for three weeks. At the end of the experimental period, various parameters of non-specific defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes and total plasma protein level were examined. Specific growth rates (SGRs) and condition factors (CFs) of the fish were also measured. Plant materials tested for immunostimulatory food additives caused an enhanced extracellular respiratory burst activity (P<0.001) compared to the control group. Especially the rainbow trout fed with a diet containing 1% aqueous extract of powdered ginger roots for three weeks exhibited a significant non-specific immune response. Phagocytosis and extracellular burst activity of blood leukocytes were significantly higher in this group than those in the control group. All plant extracts added to fish diet increased the total protein level in plasma except 0.1% ginger. The highest level of plasma proteins was observed in the group fed with 1% ginger extract containing feed.

Extracellular levels of amino acids and choline in human high grade gliomas: an intraoperative microdialysis study.

PubMed

Bianchi, L; De Micheli, E; Bricolo, A; Ballini, C; Fattori, M; Venturi, C; Pedata, F; Tipton, K F; Della Corte, L

2004-01-01

The concentrations of endogenous amino acids and choline in the extracellular fluid of human cerebral gliomas have been measured, for the first time, by in vivo microdialysis. Glioblastoma growth was associated with increased concentrations of choline, GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. There was no difference between grade III and grade IV tumors in the concentrations of phenylalanine, isoleucine, tyrosine, valine, and lysine, whereas the concentrations of choline, aspartate, taurine, GABA, leucine, and glutamate were significantly different in the two tumor-grade subgroups. In contrast to the other compounds, the concentration of glutamate was decreased in glioma. The parenchyma adjacent to the tumor showed significant changes only in the extracellular concentration of glutamate, isoleucine, and valine. The concentrations of choline and the amino acids, glutamate, leucine, taurine, and tyrosine showed significant positive correlations with the degree of cell proliferation. Epilepsy, which is relatively common in subjects with gliomas, was shown to be a significant confounding variable when the extracellular concentrations of aspartate, glutamate and GABA were considered.

Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

PubMed

Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

2015-01-01

PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices.

PubMed

Sperlágh, B; Zsilla, G; Baranyi, M; Kékes-Szabó, A; Vizi, E S

1997-10-01

The presynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 microM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 microM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found. In summary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

PubMed

Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

2015-01-01

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

Extracellular control of intracellular drug release for enhanced safety of anti-cancer chemotherapy

NASA Astrophysics Data System (ADS)

Zhu, Qian; Qi, Haixia; Long, Ziyan; Liu, Shang; Huang, Zhen; Zhang, Junfeng; Wang, Chunming; Dong, Lei

2016-06-01

The difficulty of controlling drug release at an intracellular level remains a key challenge for maximising drug safety and efficacy. We demonstrate herein a new, efficient and convenient approach to extracellularly control the intracellular release of doxorubicin (DOX), by designing a delivery system that harnesses the interactions between the system and a particular set of cellular machinery. By simply adding a small-molecule chemical into the cell medium, we could lower the release rate of DOX in the cytosol, and thereby increase its accumulation in the nuclei while decreasing its presence at mitochondria. Delivery of DOX with this system effectively prevented DOX-induced mitochondria damage that is the main mechanism of its toxicity, while exerting the maximum efficacy of this anti-cancer chemotherapeutic agent. The present study sheds light on the design of drug delivery systems for extracellular control of intracellular drug delivery, with immediate therapeutic implications.

LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus.

PubMed

Wang, Q; Liu, Q; Ma, Y; Rui, H; Zhang, Y

2007-11-01

To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either sigma(54) dependent or independent manners. The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and sigma(54) as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis.

Comparison of changes in the extracellular concentration of noradrenaline in rat frontal cortex induced by sibutramine or d-amphetamine: modulation by α2-adrenoceptors

PubMed Central

Wortley, K E; Hughes, Z A; Heal, D J; Stanford, S C

1999-01-01

The effects of sibutramine (0.25–10 mg kg−1, i.p.) on extracellular noradrenaline concentration in the frontal cortex of halothane-anaesthetized rats were compared with those of d-amphetamine (1–3 mg kg−1, i.p.) using in vivo microdialysis. The role of presynaptic α2-adrenoceptors in modulating the effects of these drugs on extracellular noradrenaline concentration were also investigated by pretreating rats with the selective α2-adrenoceptor antagonist, RX821002.Sibutramine induced a gradual and sustained increase in extracellular noradrenaline concentration. The dose-response relationship was described by a bell-shaped curve with a maximum effect at 0.5 mg kg−1. In contrast, d-amphetamine induced a rapid increase in extracellular noradrenaline concentration, the magnitude of which paralleled drug dose.Pretreatment with the α2-adrenoceptor antagonist, RX821002 (dose 3 mg kg−1, i.p.) increased by 5 fold the accumulation of extracellular noradrenaline caused by sibutramine (10 mg kg−1) and reduced the latency of sibutramine to reach its maximum effect from 144–56 min.RX821002-pretreatment increased by only 2.5 fold the increase in extracellular noradrenaline concentration caused by d-amphetamine alone (10 mg kg−1) and had no effect on the latency to reach maximum.These findings support evidence that sibutramine acts as a noradrenaline uptake inhibitor in vivo and that the effects of this drug are blunted by indirect activation of presynaptic α2-adreno-ceptors. In contrast, the rapid increase in extracellular noradrenaline concentration induced by d-amphetamine is consistent with this being mainly due to an increase in Ca2+-independent release of noradrenaline. PMID:10482917

Brain Hyperglycemia Induced by Heroin: Association with Metabolic Neural Activation.

PubMed

Solis, Ernesto; Bola, R Aaron; Fasulo, Bradley J; Kiyatkin, Eugene A

2017-02-15

Glucose enters the brain extracellular space from arterial blood, and its proper delivery is essential for metabolic activity of brain cells. By using enzyme-based biosensors coupled with high-speed amperometry in freely moving rats, we previously showed that glucose levels in the nucleus accumbens (NAc) display high variability, increasing rapidly following exposure to various arousing stimuli. In this study, the same technology was used to assess NAc glucose fluctuations induced by intravenous heroin. Heroin passively injected at a low dose optimal for maintaining self-administration behavior (100 μg/kg) induces a rapid but moderate glucose rise (∼150-200 μM or ∼15-25% over resting baseline). When the heroin dose was doubled and tripled, the increase became progressively larger in magnitude and longer in duration. Heroin-induced glucose increases also occurred in other brain structures (medial thalamus, lateral striatum, hippocampus), suggesting that brain hyperglycemia is a whole-brain phenomenon but changes were notably distinct in each structure. While local vasodilation appears to be the possible mechanism underlying the rapid rise in extracellular glucose levels, the driving factor for this vasodilation (central vs peripheral) remains to be clarified. The heroin-induced NAc glucose increases positively correlated with increases in intracerebral heat production determined in separate experiments using multisite temperature recordings (NAc, temporal muscle and skin). However, glucose levels rise very rapidly, preceding much slower increases in brain heat production, a measure of metabolic activation associated with glucose consumption.

Dynamic Changes in Cervical Glycosaminoglycan Composition during Normal Pregnancy and Preterm Birth

PubMed Central

Akgul, Yucel; Holt, Roxane; Mummert, Mark; Word, Ann

2012-01-01

Glycosaminoglycans (GAG) have diverse functions that regulate macromolecular assembly in the extracellular matrix. During pregnancy, the rigid cervix transforms to a pliable structure to allow birth. Quantitative assessment of cervical GAG is a prerequisite to identify GAG functions in term and preterm birth. In the current study, total GAG levels increased at term, yet the abundance, chain length, and sulfation levels of sulfated GAG remained constant. The increase in total GAG resulted exclusively from an increase in hyaluronan (HA). HA can form large structures that promote increased viscosity, hydration, and matrix disorganization as well as small structures that have roles in inflammation. HA levels increased from 19% of total GAG in early pregnancy to 71% at term. Activity of the HA-metabolizing enzyme, hyaluronidase, increased in labor, resulting in metabolism of large to small HA. Similar to mice, HA transitions from high to low molecular weight in term human cervix. Mouse preterm models were also characterized by an increase in HA resulting from differential expression of the HA synthase (Has) genes, with increased Has1 in preterm in contrast to Has2 induction at term. The Has2 gene but not Has1 is regulated in part by estrogen. These studies identify a shift in sulfated GAG dominance in the early pregnant cervix to HA dominance in term and preterm ripening. Increased HA synthesis along with hyaluronidase-induced changes in HA size in mice and women suggest diverse contributions of HA to macromolecular changes in the extracellular matrix, resulting in loss of tensile strength during parturition. PMID:22529214

Effects of the neurotoxin MPTP and pargyline protection on extracellular energy metabolites and dopamine levels in the striatum of freely moving rats.

PubMed

Bazzu, Gianfranco; Rocchitta, Gaia; Migheli, Rossana; Alvau, Maria Domenica; Zinellu, Manuel; Puggioni, Giulia; Calia, Giammario; Mercanti, Giulia; Giusti, Pietro; Desole, Maria Speranza; Serra, Pier Andrea

2013-11-13

The neurotoxin MPTP is known to induce dopamine release and depletion of ATP in the striatum of rats. Therefore, we studied the changes induced by MPTP and pargyline protection both on striatal dopamine release and on extracellular energy metabolites in freely moving rats, using dual asymmetric-flow microdialysis. A dual microdialysis probe was inserted in the right striatum of rats. MPTP (25mg/kg, 15mg/kg, 10mg/kg) was intraperitoneally administered for three consecutive days. MAO-B inhibitor pargyline (15mg/kg) was systemically administered before neurotoxin administration. The first MPTP dose induced an increase in dialysate dopamine and a decrease of DOPAC levels in striatal dialysate. After the first neurotoxin administration, increases in striatal glucose, lactate, pyruvate, lactate/pyruvate (L/P) and lactate/glucose (L/G) ratios were observed. Subsequent MPTP administrations showed a progressive reduction of dopamine, glucose and pyruvate levels with a concomitant further increase in lactate levels and L/P and L/G ratios. At day 1, pargyline pre-treatment attenuated the MPTP-induced changes in all studied analytes. Starting from day 2, pargyline prevented the depletion of dopamine, glucose and pyruvate while reduced the increase of lactate, L/P ratio and L/G ratio. These in vivo results suggest a pargyline neuroprotection role against the MPTP-induced energetic impairment consequent to mitochondrial damage. This neuroprotective effect was confirmed by TH immunostaining of the substantia nigra. © 2013 Elsevier B.V. All rights reserved.

Melanin production through novel processing of proopiomelanocortin in the extracellular compartment of the auricular skin of C57BL/6 mice after UV-irradiation.

PubMed

Yamamoto, Hiroyuki; Yamane, Tomohiro; Iguchi, Kazuaki; Tanaka, Kiyotaka; Iddamalgoda, Arunasiri; Unno, Keiko; Hoshino, Minoru; Takeda, Atsushi

2015-09-29

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1-8). Furthermore, ACTH(1-8) competed with [(125)I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1-8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1-8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

Evaluating removal of microcystin-LR from drinking water using Granular Activated Carbon with various amounts of NOM Preloading

EPA Science Inventory

Increases in cyanotoxin containing harmful algal blooms in recent years has raised the level of concern within the water treatment industry to remove these dangerous bacteria and chemicals. Typical methods used to treat intracellular and extracellular cyanotoxins include pretreat...

Using Rapid Small Scale Column Testing to Evaluate Granular Activated Carbon Adsorption of Cyanotoxins from Drinking Water

EPA Science Inventory

Increases in cyanotoxin containing harmful algal blooms in recent years has raised the level of concern within the water treatment industry to remove these dangerous bacteria and chemicals. Typical methods used to treat intracellular and extracellular cyanotoxins include pretreat...

Commercial levels of chymosin production by Aspergillus.

PubMed

Dunn-Coleman, N S; Bloebaum, P; Berka, R M; Bodie, E; Robinson, N; Armstrong, G; Ward, M; Przetak, M; Carter, G L; LaCost, R

1991-10-01

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Characterization of intracellular and extracellular saxitoxin levels in both field and cultured Alexandrium spp. samples from Sequim Bay, Washington.

PubMed

Lefebvre, Kathi A; Bill, Brian D; Erickson, Aleta; Baugh, Keri A; O'Rourke, Lohna; Costa, Pedro R; Nance, Shelly; Trainer, Vera L

2008-05-14

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004-2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 +/- 9.7% of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

Characterization of Intracellular and Extracellular Saxitoxin Levels in Both Field and Cultured Alexandrium spp. Samples from Sequim Bay, Washington

PubMed Central

Lefebvre, Kathi A.; Bill, Brian D.; Erickson, Aleta; Baugh, Keri A.; O’Rourke, Lohna; Costa, Pedro R.; Nance, Shelly; Trainer, Vera L.

2008-01-01

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004–2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region. PMID:18728762

Restoration of long-term potentiation in middle-aged hippocampus after induction of brain-derived neurotrophic factor.

PubMed

Rex, Christopher S; Lauterborn, Julie C; Lin, Ching-Yi; Kramár, Eniko A; Rogers, Gary A; Gall, Christine M; Lynch, Gary

2006-08-01

Restoration of neuronal viability and synaptic plasticity through increased trophic support is widely regarded as a potential therapy for the cognitive declines that characterize aging. Previous studies have shown that in the hippocampal CA1 basal dendritic field deficits in the stabilization of long-term potentiation (LTP) are evident by middle age. The present study tested whether increasing endogenous brain-derived neurotrophic factor (BDNF) could reverse this age-related change. We report here that in middle-aged (8- to 10-mo-old) rats, in vivo treatments with a positive AMPA-type glutamate receptor modulator both increase BDNF protein levels in the cortical telencephalon and restore stabilization of basal dendritic LTP as assessed in acute hippocampal slices 18 h after the last drug treatment. These effects were not attributed to enhanced synaptic transmission or to facilitation of burst responses used to induce LTP. Increasing extracellular levels of BDNF by exogenous application to slices of middle-aged rats was also sufficient to rescue the stabilization of basal dendritic LTP. Finally, otherwise stable LTP in ampakine-treated middle-aged rats can be eliminated by infusion of the extracellular BDNF scavenger TrkB-Fc. Together these results indicate that increases in endogenous BDNF signaling can offset deficits in the postinduction processes that stabilize LTP.

Fluoxetine and WAY 100,635 dissociate increases in scototaxis and analgesia induced by conspecific alarm substance in zebrafish (Danio rerio Hamilton 1822).

PubMed

Maximino, Caio; Lima, Monica Gomes; Costa, Carina Cardoso; Guedes, Iêda Maria Louzada; Herculano, Anderson Manoel

2014-09-01

Alarm reactions to a substance secreted by the damaged skin of conspecifics and closely-related species are increasingly being recognized as fear-like responses in fish. The neurochemical underpinnings of these effects are so far unknown; however, given the role of the serotonergic system on defensive behavior, it is possible that the alarm reaction is mediated by this monoamine. Exposure to conspecific alarm substance (CAS) increased anxiety-like behavior in the light/dark test in zebrafish and decreased nocifensive behavior. These effects were accompanied by increases in blood glucose, hemoglobin, epinephrine and norepinephrine levels, as well as extracellular levels of serotonin in the brain. Pretreatment with fluoxetine blocked the anxiogenic effects of CAS on the light/dark test as well as all physiological parameters and the increase in extracellular brain 5-HT, but not the reduction in nocifensive behavior. Conversely, pretreatment with the 5-HT1AR antagonist WAY 100635 blocked the effects on nocifensive behavior, but not the effects on anxiety-like behavior nor on physiological parameters. These results point to an important and complex role of the serotonergic system in the mediation of fear-potentiated behavior in the light/dark test and in fear-induced analgesia in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of antioxidant enzymes in Ectocarpus siliculosus.

PubMed

González, Alberto; Sáez, Claudio A; Morales, Bernardo; Moenne, Alejandra

2018-05-01

The existence of functional Transient Receptor Potential (TRP) channels was analyzed in Ectocarpus siliculosus using agonists of human TRPs and specific antagonists of TRPA1, TRPC5, TRPM8 and TRPV; intracellular calcium was detected for 60 min. Increases in intracellular calcium were observed at 13, 29, 39 and 50-52 min, which appeared to be mediated by the activation of TRPM8/V1 at 13 min, TRPV1 at 29 min, TRPA1/V1 at 39 min and TRPA1/C5 at 50-52 min. In addition, intracellular calcium increases appear to be due to extracellular calcium entry, not requiring protein kinase activation. On the other hand, 2.5 μM copper exposure induced increased intracellular calcium at 13, 29, 39 and 51 min, likely due to the activation of a TRPA1/V1 at 13 min, TRPA1/C5/M8 at 29 min, TRPC5/M8 at 39 min, and a TRPC5/V1 at 51 min. The increases in intracellular calcium induced by copper were due to extracellular calcium entry and required protein kinase activation. Furthermore, from 3 to 24 h, copper exposure induced an increase in the level of transcripts encoding antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and peroxiredoxin. The described upregulation decreased with inhibitors of CaMK, PKA, PKC, PKG and CBLPK, as well as with a mixture of TRP inhibitors. Thus, copper induces the activation of TRP channels allowing extracellular calcium entry as well as the activation of CaMK, PKA, PKC, PKG and CBLPK leading to increased expression of genes encoding antioxidant enzymes in E. siliculosus. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Extracellular levels of lactate, but not oxygen, reflect sleep homeostasis in the rat cerebral cortex.

PubMed

Dash, Michael B; Tononi, Giulio; Cirelli, Chiara

2012-07-01

It is well established that brain metabolism is higher during wake and rapid eye movement (REM) sleep than in nonrapid eye movement (NREM) sleep. Most of the brain's energy is used to maintain neuronal firing and glutamatergic transmission. Recent evidence shows that cortical firing rates, extracellular glutamate levels, and markers of excitatory synaptic strength increase with time spent awake and decline throughout NREM sleep. These data imply that the metabolic cost of each behavioral state is not fixed but may reflect sleep-wake history, a possibility that is investigated in the current report. Chronic (4d) electroencephalographic (EEG) recordings in the rat cerebral cortex were coupled with fixed-potential amperometry to monitor the extracellular concentration of oxygen ([oxy]) and lactate ([lac]) on a second-by-second basis across the spontaneous sleep-wake cycle and in response to sleep deprivation. Basic sleep research laboratory. Wistar Kyoto (WKY) adult male rats. N/A. Within 30-60 sec [lac] and [oxy] progressively increased during wake and REM sleep and declined during NREM sleep (n = 10 rats/metabolite), but with several differences. [Oxy], but not [lac], increased more during wake with high motor activity and/or elevated EEG high-frequency power. Meanwhile, only the NREM decline of [lac] reflected sleep pressure as measured by slow-wave activity, mirroring previous results for cortical glutamate. The observed state-dependent changes in cortical [lac] and [oxy] are consistent with higher brain metabolism during waking and REM sleep in comparison with NREM sleep. Moreover, these data suggest that glycolytic activity, most likely through its link with glutamatergic transmission, reflects sleep homeostasis.

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

PubMed

Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

2016-01-01

Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.

PubMed

Gallagher, Ferdia A; Sladen, Helen; Kettunen, Mikko I; Serrao, Eva M; Rodrigues, Tiago B; Wright, Alan; Gill, Andrew B; McGuire, Sarah; Booth, Thomas C; Boren, Joan; McIntyre, Alan; Miller, Jodi L; Lee, Shen-Han; Honess, Davina; Day, Sam E; Hu, De-En; Howat, William J; Harris, Adrian L; Brindle, Kevin M

2015-10-01

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH. ©2015 American Association for Cancer Research.

Sources of extracellular tau and its signaling.

PubMed

Avila, Jesús; Simón, Diana; Díaz-Hernández, Miguel; Pintor, Jesús; Hernández, Félix

2014-01-01

The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

Modafinil attenuates reinstatement of cocaine seeking: role for cystine-glutamate exchange and metabotropic glutamate receptors.

PubMed

Mahler, Stephen V; Hensley-Simon, Megan; Tahsili-Fahadan, Pouya; LaLumiere, Ryan T; Thomas, Charles; Fallon, Rebecca V; Kalivas, Peter W; Aston-Jones, Gary

2014-01-01

Modafinil may be useful for treating stimulant abuse, but the mechanisms by which it acts to do so are unknown. Indeed, a primary effect of modafinil is to inhibit dopamine transport, which typically promotes rather than inhibits motivated behavior. Therefore, we examined the role of nucleus accumbens extracellular glutamate and the group II metabotropic glutamate receptor (mGluR2/3) in modafinil effects. One group of rats was trained to self-administer cocaine for 10 days and extinguished, then given priming injections of cocaine to elicit reinstatement. Modafinil (300 mg/kg, intraperitoneal) inhibited reinstated cocaine seeking (but did not alter extinction responding by itself), and this effect was prevented by pre-treatment with bilateral microinjections of the mGluR2/3 antagonist LY-341495 (LY) into nucleus accumbens core. No reversal of modafinil effects was seen after unilateral accumbens core LY, or bilateral LY in the rostral pole of accumbens. Next, we sought to explore effects of modafinil on extracellular glutamate levels in accumbens after chronic cocaine. Separate rats were administered non-contingent cocaine, and after 3 weeks of withdrawal underwent accumbens microdialysis. Modafinil increased extracellular accumbens glutamate in chronic cocaine, but not chronic saline-pre-treated animals. This increase was prevented by reverse dialysis of cystine-glutamate exchange or voltage-dependent calcium channel antagonists. Voltage-dependent sodium channel blockade partly attenuated the increase in glutamate, but mGluR1 blockade did not. We conclude that modafinil increases extracellular glutamate in nucleus accumbens from glial and neuronal sources in cocaine-exposed rats, which may be important for its mGluR2/3-mediated antirelapse properties. © 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

PubMed Central

Gaul, Daniel S; Weber, Julien; van Tits, Lambertus J; Sluka, Susanna; Pasterk, Lisa; Reiner, Martin F; Calatayud, Natacha; Lohmann, Christine; Klingenberg, Roland; Pahla, Jürgen; Vdovenko, Daria; Tanner, Felix C; Camici, Giovanni G; Eriksson, Urs; Auwerx, Johan; Mach, François; Windecker, Stephan; Rodondi, Nicolas; Lüscher, Thomas F; Winnik, Stephan; Matter, Christian M

2018-01-01

Abstract Aims Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke. PMID:29444200

Protective Effects of Extracellular and Intracellular Polysaccharides on Hepatotoxicity by Hericium erinaceus SG-02.

PubMed

Cui, Fangyuan; Gao, Xia; Zhang, Jianjun; Liu, Min; Zhang, Chen; Xu, Nuo; Zhao, Huajie; Lin, Lin; Zhou, Meng; Jia, Le

2016-09-01

The protective effects of extracellular and intracellular polysaccharides from Hericium erinaceus SG-02 on the CCl4-induced hepatic injury of mice were investigated in this work. By the analysis of GC, the extracellular polysaccharides (EPS) were composed of arabinose, mannose, galactose, and glucose with a ratio of 1:7:14:52, and the composition of intracellular polysaccharides (IPS) was rhamnose, xylose, mannose, galactose, and glucose with a ratio of 3:4:7:14:137. The model of hepatic injury of mice was induced by CCl4 and three tested levels (200, 400, and 800 mg/kg) of EPS and IPS were set as the experimental group. Results showed that the aspartate aminotransferase and glutamic pyruvic transaminase activities in serum were reduced by the supplement of EPS and IPS, while the blood lipid levels including cholesterol, triglyceride, and albumin were improved. In liver tissue, the lipid peroxidation and malondialdehyde were largely decreased, and the superoxide dismutase and catalase activities were significantly increased. The evidence demonstrated that the EPS and IPS of H. erinaceus SG-02 were protective for liver injury. The histopathological observations of mice liver slices indicated that EPS and IPS had obvious effects on liver protection.

Hyperpalatable Diet and Physical Exercise Modulate the Expression of the Glial Monocarboxylate Transporters MCT1 and 4.

PubMed

Portela, Luis V; Brochier, Andressa W; Haas, Clarissa B; de Carvalho, Afonso Kopczynski; Gnoato, Jussania A; Zimmer, Eduardo R; Kalinine, Eduardo; Pellerin, Luc; Muller, Alexandre P

2017-10-01

Hyperpalatable diets (HP) impair brain metabolism, and regular physical exercise has an apparent opposite effect. Here, we combined a prior long-term exposure to HP diet followed by physical exercise and evaluated the impact on some neuroenergetic components and on cognitive performance. We assessed the extracellular lactate concentration, expression of monocarboxylate transporters (MCTs), pyruvate dehydrogenase (PDH), and mitochondrial function in the hippocampus. Male C57BL/6J mice were fed 4 months with HP or a control diet. Subsequently, they were divided in the following groups: control diet sedentary (CDS), control diet exercise (CDE), HP diet sedentary (HPS), and HP diet exercise (HPE) (n = 15 per group) and were engaged for an additional 30-day period of voluntary exercise and HP diet. Relative to the control situation, exercise increased MCT1, MCT4, and PDH protein levels, while the HP diet increased MCT1 and MCT4 protein levels. The production of hydrogen peroxide (H 2 O 2 ) and the mitochondrial membrane potential (∆Ѱ m ) stimulated by succinate in hippocampal homogenates were not significantly different between groups. ADP phosphorylation and the maximal respiratory rate induced by FCCP showed similar responses between groups, implying a normal mitochondrial function. Also, extracellular brain lactate levels were increased in the HPE group compared to other groups soon after performing the Y-maze task. However, such enhanced lactate levels were not associated with improved memory performance. In summary, hippocampal protein expression levels of MCT1 and 4 were increased by physical exercise and HP diet, whereas PDH was only increased by exercise. These observations indicate that a hippocampal metabolic reprogramming takes place in response to these environmental factors.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

PubMed Central

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

PubMed

Ichikawa, Shoji; Gerard-O'Riley, Rita L; Acton, Dena; McQueen, Amie K; Strobel, Isabel E; Witcher, Phillip C; Feng, Jian Q; Econs, Michael J

2017-03-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. Copyright © 2017 by the Endocrine Society.

The atypical antipsychotic quetiapine increases both noradrenaline and dopamine release in the rat prefrontal cortex.

PubMed

Pira, Luigi; Mongeau, Raymond; Pani, Luca

2004-11-03

Quetiapine is a novel atypical antipsychotic drug with multi-receptorial affinity. Using in vivo microdialysis, we investigated if quetiapine modulates extracellular noradrenaline and dopamine in brain areas generally believed to be involved in the pathophysiology of schizophrenia and in the action of antipsychotic drugs. Quetiapine (5, 10 and 20 mg/kg, i.p.) increased levels of noradrenaline in both the prefrontal cortex and the caudate nucleus, while it increased dopamine levels mainly in the prefrontal cortex. It is argued that the marked increase of dopaminergic transmission in the prefrontal cortex induced by quetiapine might be relevant to its therapeutical action.

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells.

PubMed

Procino, Giuseppe; Carmosino, Monica; Tamma, Grazia; Gouraud, Sabine; Laera, Antonia; Riccardi, Daniela; Svelto, Maria; Valenti, Giovanna

2004-12-01

Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.

Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart.

PubMed

Luo, Hongmin; Sawashita, Jinko; Tian, Geng; Liu, Yingye; Li, Lin; Ding, Xin; Xu, Zhe; Yang, Mu; Miyahara, Hiroki; Mori, Masayuki; Qian, Jinze; Wang, Yaoyong; Higuchi, Keiichi

2015-03-01

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2(c) mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.

Increased tenascin C and Toll-like receptor 4 levels in visceral adipose tissue as a link between inflammation and extracellular matrix remodeling in obesity.

PubMed

Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Rotellar, Fernando; Valentí, Victor; Silva, Camilo; Gil, María J; Salvador, Javier; Frühbeck, Gema

2012-10-01

Obesity is associated with an altered inflammatory and extracellular matrix (ECM) profile. Tenascin C (TNC) is an ECM glycoprotein with proinflammatory effects. We aimed to explore the expression levels of TNC in adipose tissue analyzing the contribution of adipocytes and stromovascular fraction cells (SVFC) as well as its impact on inflammation and ECM regulation. We also analyzed the effect of the stimulation with TNF-α and lipopolysaccharide (LPS) on both SVFC and adipocytes. Samples obtained from 75 subjects were used in the study. Expression levels of TNC, TLR4, MMP2, and MMP9 were analyzed in visceral adipose tissue (VAT) as well as in both adipocytes and SVFC. In addition, Tnc expression was measured in two mice models of obesity. We show, for the first time, that VAT expression levels of TNC are increased in normoglycemic and type 2 diabetic obese patients (P<0.01) as well as in obese patients with nonalcoholic steatohepatitis (P<0.01). Furthermore, expression levels of Tnc in epididymal adipose tissue from two different mice models of obesity were significantly increased (P<0.01). TNC and TLR4 were mainly expressed by SVFC, and its expression was significantly enhanced (P<0.01) by TNF-α treatment. LPS treatment also increased mRNA levels of TNC. Moreover, the addition of exogenous TNC induced (P<0.05) TLR4 and CCL2 mRNA expression in human adipocyte cultures. These findings indicate that TNC is involved in the etiopathology of obesity via visceral adipose tissue inflammation representing a link with ECM remodeling.

Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

PubMed

Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

2015-10-01

Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xi, E-mail: Xi.Yang@fda.hhs.gov; Salminen, William F., E-mail: Willie.Salminen@parexel.com; Shi, Qiang, E-mail: Qiang.Shi@fda.hhs.gov

Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n = 10), 2) hospitalized children receiving therapeutic doses of APAP (n = 10) and 3) children hospitalized for APAP overdose (n = 8). Out of 147 miRNAs detected in themore » APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R = 0.94; p < 0.01) followed by miR-375 (R = 0.70; p = 0.05). Conclusion: Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity. - Highlights: • Serum miR-122 and miR-375 levels were increased in children with APAP overdose. • Urine levels of miR-375 and miR-940 were increased in the APAP overdose group. • Peak serum miR-122 levels were correlated with peak serum APAP protein adducts.« less

Differential modulation of degradative and repair responses of interleukin-1-treated chondrocytes by platelet-derived growth factor.

PubMed Central

Harvey, A K; Stack, S T; Chandrasekhar, S

1993-01-01

Interleukin 1 (IL-1) plays a dual role in cartilage matrix degeneration by promoting extracellular proteinase action such as the matrix metalloproteinases (increased degradation) and by suppressing the synthesis of extracellular matrix molecules (inhibition of repair). Platelet-derived growth factor (PDGF) is a wound-healing hormone which is released along with IL-1 during the inflammatory response. Since previous studies have shown that PDGF enhances IL-1 alpha effects on metalloproteinase activity, in this report, we have examined whether PDGF modifies IL-1 beta effects on cartilage proteoglycan synthesis. Initially, we confirmed that rabbit articular chondrocytes treated with IL-1 beta + PDGF induced higher proteinase activity, in comparison with IL-1-treated cells. We further observed that the increased proteinase activity correlated with an increase in the synthesis of collagenase/stromelysin proteins and a corresponding increase in the steady-state mRNA levels for both the enzymes. Studies on IL-1 receptor expression suggested that PDGF caused an increase in IL-1 receptor expression which, by augmenting the IL-1 response, may have led to the increase in proteinase induction. Analysis of proteoglycan synthesis confirmed that IL-1 reduced the incorporation of sulphated proteoglycan, aggrecan, into the extracellular matrix of chondrocytes, whereas PDGF stimulated it. However, cells treated with IL-1 + PDGF synthesized normal levels of aggrecan. This is in contrast with cells treated with IL-1 + fibroblast growth factor, in which case only proteinase activity was potentiated. The results allow us to conclude that (a) the two effector functions that play a role in matrix remodelling, namely matrix lysis (proteinase induction) and matrix repair (proteoglycan synthesis), occur via distinct pathways and (b) PDGF may play a crucial role in cartilage repair by initially causing matrix degradation followed by promoting new matrix synthesis. Images Figure 1 Figure 2 Figure 5 Figure 6 PMID:8503839

Changes in Acetylcholine Extracellular Levels during Cognitive Processes

ERIC Educational Resources Information Center

Pepeu, Giancarlo; Giovannini, Maria Grazia

2004-01-01

Measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered a tool for identifying the neuronal systems involved in specific behavioral responses or cognitive processes. Acetylcholine (ACh) is the first neurotransmitter whose diffusion from the central nervous system was investigated and whose extracellular…

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

PubMed Central

Schiro, Faith R.; Pajor, Ana M.; Hamm, L. Lee

2011-01-01

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na+-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation. PMID:21123491

[In vitro evaluation of cutaneous allergic reaction induced by chemicals using dendritic cells].

PubMed

Zhang, Yu-bin; Lin, Hui-fen; Lv, Luo; Hua, Wei-guang; Tian, Fang; Shen, Guang-zu; Xia, Zhao-lin; Jin, Xi-peng

2008-03-01

To investigate the use of dendritic cells derived from mice bone marrow to evaluate the cutaneous allergic reaction induced by chemical sensitizers. Dendritic cells derived from mice bone marrow were cultured and administrated with 2, 4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4), sodium dodecyl sulfate (SDS) and hexyl cinnamic aldehyde (HCA), respectively. Cell membrane molecule CD86 and extracellular IL-1 beta, IL-6 and IL-12 were detected after 0, 1, 6, 12, 24, 36, 48 hour's administration, respectively. CD86 expression reached the highest level after exposure to DNCB for 48 h, and increased by about 279% compared with the control (P < 0.05), while it was lower than that of control after administrated with NiSO4 and HCA for 1 h and 6 h, and SDS for 36 h, respectively (P < 0.05). Extracellular IL-1 beta increased greatly after exposure to NiSO4 just for 1 h, with the maximum at 48 h (298 pg/ml, P < 0.05), and after exposure to HCA for 6 h, with maximum at 48 h (84 pg/ml, P < 0.05). However, it didn't fluctuate significantly after administrated with DNCB and SDS respectively, compared with the control. Extracellular IL-6 increased significantly after exposure to NiSO4 for 1 h, with the maximum at 24 h (2152 pg/ml, P < 0.05). After exposure to HCA, extracellular IL-6 reached the maximum at 1 h (1403 pg/ml), and then it was decreased quickly, but still higher than the control (P < 0.05), while it didn't change significantly after treatment with DNCB and SDS, compared with the control (P > 0.05). Extracellular IL-12 was not detected out among all the groups. Chemical sensitizer DNCB could induce the high expression of CD86 on DC membrane, and NiSO4 and HCA could induce DC to release IL-1 beta and IL-6. However, the irritant SDS had no such effect.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

PubMed Central

Díaz-Vegas, Alexis; Campos, Cristian A.; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

2015-01-01

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

Baicalein, a Component of Scutellaria baicalensis, Attenuates Kidney Injury Induced by Myocardial Ischemia and Reperfusion.

PubMed

Lai, Chang-Chi; Huang, Po-Hsung; Yang, An-Han; Chiang, Shu-Chiung; Tang, Chia-Yu; Tseng, Kuo-Wei; Huang, Cheng-Hsiung

2016-02-01

Acute kidney injury is a common and severe complication of acute myocardial infarction and cardiac surgery. It results in increased mortality, morbidity, and duration of hospitalization. Baicalein is a component of the root of Scutellaria baicalensis, which has traditionally been used to treat cardiovascular and liver diseases in Asia. In this study, we investigated whether baicalein can attenuate kidney injury induced by myocardial ischemia and reperfusion in rats. Myocardial ischemia and reperfusion, induced by a 40-minute occlusion and a 3-hour reperfusion of the left anterior descending coronary artery, significantly increased blood urea nitrogen and creatinine levels in addition to causing histological changes in the kidneys. Kidney apoptosis was also significantly increased. Furthermore, myocardial ischemia and reperfusion significantly increased the serum levels of tumor necrosis factor-α, interleukin-1, and interleukin-6 as well as the tumor necrosis factor-α levels in the kidneys. Intravenous pretreatment with baicalein (in doses of 3, 10, or 30 mg/kg), however, significantly reduced the increases in the creatinine level, renal histological damage, and apoptosis induced by myocardial ischemia and reperfusion. In addition, the increases in the serum levels of tumor necrosis factor-α, interleukin-1, and interleukin-6, and of tumor necrosis factor-α in the kidneys were significantly reduced. Western blot analysis revealed that baicalein significantly increased Bcl-2 and reduced Bax in the kidneys. The phosphorylation of Akt and extracellular signal-regulated kinases 1 and 2 was also significantly increased. In conclusion, baicalein significantly attenuates kidney injury induced by myocardial ischemia and reperfusion. The underlying mechanisms might be related to the inhibition of apoptosis, possibly through the reduction of tumor necrosis factor-α production, the modulation of Bcl-2 and Bax, and the activation of Akt and extracellular signal-regulated kinases 1 and 2. Georg Thieme Verlag KG Stuttgart · New York.

Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP

PubMed Central

Vardjan, Nina; Chowdhury, Helena H.; Horvat, Anemari; Velebit, Jelena; Malnar, Maja; Muhič, Marko; Kreft, Marko; Krivec, Špela G.; Bobnar, Saša T.; Miš, Katarina; Pirkmajer, Sergej; Offermanns, Stefan; Henriksen, Gjermund; Storm-Mathisen, Jon; Bergersen, Linda H.; Zorec, Robert

2018-01-01

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism. PMID:29867342

The extracellular domain of neurotrophin receptor p75 as a candidate biomarker for amyotrophic lateral sclerosis.

PubMed

Shepheard, Stephanie R; Chataway, Tim; Schultz, David W; Rush, Robert A; Rogers, Mary-Louise

2014-01-01

Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1(G93A) mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson's disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTR(ECD) levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1(G93A) mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.

Extracellular adenosine triphosphate affects systemic and kidney immune cell populations in pregnant rats.

PubMed

Spaans, Floor; Melgert, Barbro N; Borghuis, Theo; Klok, Pieter A; de Vos, Paul; Bakker, Winston W; van Goor, Harry; Faas, Marijke M

2014-09-01

Changes in the systemic immune response are found in preeclampsia. This may be related to high extracellular adenosine triphosphate (ATP) levels. The question arose whether ATP could affect immune responses in pregnancy. Previously, we investigated whether ATP affected monocyte activation and subpopulations. Here, we investigated ATP-induced changes in other immune cell populations in pregnant rats, systemically and in the kidney, an affected organ in preeclampsia. Using flow cytometry or immunohistochemistry, blood and kidney leukocytes were studied in pregnant and non-pregnant rats at different intervals after ATP or saline infusion. Adenosine triphosphate (ATP) infusion induced increased peripheral blood non-classical monocytes and decreased T lymphocyte subsets in pregnant rats only, higher glomerular macrophage and T lymphocyte numbers in non-pregnant animals 1 day after infusion, and higher glomerular macrophage numbers in pregnant rats 6 days after infusion. Adenosine triphosphate (ATP) infusion in pregnant rats induced a pregnancy-specific inflammatory response. Increased ATP levels could potentially contribute to development of the inflammatory response of preeclampsia. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pirfenidone normalizes the tumor microenvironment to improve chemotherapy

PubMed Central

Papageorgis, Panagiotis; Voutouri, Chrysovalantis; Stylianopoulos, Triantafyllos

2017-01-01

Normalization of the tumor microenvironment by selectively targeting components of the tumor extracellular matrix has been recently proposed to have the potential to decompress tumor blood vessels, increase vessel perfusion and thus, improve drug delivery and the efficacy of cancer therapy. Therefore, we now need to identify safe and well tolerated pharmaceutical agents that are able to remodel the microenvironment of solid tumors and enhance chemotherapy. In this study, we repurposed Pirfenidone, a clinically approved anti-fibrotic drug for the treatment of idiopathic pulmonary fibrosis, to investigate its possible role on tumor microenvironment normalization. Using two orthotopic mammary tumor models we demonstrate that Pirfenidone reduces collagen and hyaluronan levels and, as a result, significantly increases blood vessel functionality and perfusion and improves the anti-tumor efficacy of doxorubicin. Reduction of extracellular matrix components were mediated via TGFβ signaling pathway inhibition due to downregulation of TGFβ1, COL1A1, COL3A1, HAS2, HAS3 expression levels. Our findings provide evidence that repurposing Pirfenidone could be used as a promising strategy to enhance drug delivery to solid tumors by normalizing the tumor microenvironment. PMID:28445938

Modeling the mechanics of cells in the cell-spreading process driven by traction forces

NASA Astrophysics Data System (ADS)

Fang, Yuqiang; Lai, King W. C.

2016-04-01

Mechanical properties of cells and their mechanical interaction with the extracellular environments are main factors influencing cellular function, thus indicating the progression of cells in different disease states. By considering the mechanical interactions between cell adhesion molecules and the extracellular environment, we developed a cell mechanical model that can characterize the mechanical changes in cells during cell spreading. A cell model was established that consisted of various main subcellular components, including cortical cytoskeleton, nuclear envelope, actin filaments, intermediate filaments, and microtubules. We demonstrated the structural changes in subcellular components and the changes in spreading areas during cell spreading driven by traction forces. The simulation of nanoindentation tests was conducted by integrating the indenting force to the cell model. The force-indentation curve of the cells at different spreading states was simulated, and the results showed that cell stiffness increased with increasing traction forces, which were consistent with the experimental results. The proposed cell mechanical model provides a strategy to investigate the mechanical interactions of cells with the extracellular environments through the adhesion molecules and to reveal the cell mechanical properties at the subcellular level as cells shift from the suspended state to the adherent state.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

PubMed

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Modeling the mechanics of cells in the cell-spreading process driven by traction forces.

PubMed

Fang, Yuqiang; Lai, King W C

2016-04-01

Mechanical properties of cells and their mechanical interaction with the extracellular environments are main factors influencing cellular function, thus indicating the progression of cells in different disease states. By considering the mechanical interactions between cell adhesion molecules and the extracellular environment, we developed a cell mechanical model that can characterize the mechanical changes in cells during cell spreading. A cell model was established that consisted of various main subcellular components, including cortical cytoskeleton, nuclear envelope, actin filaments, intermediate filaments, and microtubules. We demonstrated the structural changes in subcellular components and the changes in spreading areas during cell spreading driven by traction forces. The simulation of nanoindentation tests was conducted by integrating the indenting force to the cell model. The force-indentation curve of the cells at different spreading states was simulated, and the results showed that cell stiffness increased with increasing traction forces, which were consistent with the experimental results. The proposed cell mechanical model provides a strategy to investigate the mechanical interactions of cells with the extracellular environments through the adhesion molecules and to reveal the cell mechanical properties at the subcellular level as cells shift from the suspended state to the adherent state.

Energy metabolism of intervertebral disc under mechanical loading.

PubMed

Wang, Chong; Gonzales, Silvia; Levene, Howard; Gu, Weiyong; Huang, Chun-Yuh Charles

2013-11-01

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism. © 2013 Orthopaedic Research Society.

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

PubMed

Wang, Dongmao; Mohammad, Mardhiah; Wang, Yanyan; Tan, Rachel; Murray, Lydia S; Ricardo, Sharon; Dagher, Hayat; van Agtmael, Tom; Savige, Judy

2017-07-01

X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL 2 ) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 ( P  = 0.01 and P  = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels ( P  < 0.01), and reduced ER size ( P  < 0.01 by EM and P  < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P  < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only ( P  = 0.06). Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

Neuroprotective effects of angiotensin II type 1 receptor (AT1-R) blocker via modulating AT1-R signaling and decreased extracellular glutamate levels.

PubMed

Fujita, Tomoyoshi; Hirooka, Kazuyuki; Nakamura, Takehiro; Itano, Toshifumi; Nishiyama, Akira; Nagai, Yukiko; Shiraga, Fumio

2012-06-26

To investigate the mechanism of the neuroprotective effects of the angiotensin II type 1 receptor (AT1-R) blocker against retinal ischemia-reperfusion injury in the rat. Retinal ischemia was induced by increasing intraocular pressure. Glutamate release from the rat retina and intravitreal PO(2) (partial pressure of oxygen) profiles were monitored during and after ischemia using a microdialysis biosensor and oxygen-sensitive microelectrodes. ELISA was used to measure changes in the expression of AT1-R. Retinal mRNA expressions of p47phox and p67phox were measured by real-time polymerase chain reaction. Reactive oxygen species (ROS) were measured using dihydroethidium. Administration of candesartan, which is an AT1-R blocker (ARB), suppressed ischemia-induced increases in the extracellular glutamate. Candesartan also attenuated the increase in intravitreal PO(2) during reperfusion. AT1-R expression peaked at 12 hours after reperfusion. Although there was an increase in the retinal mRNA expression of p47phox and p64phox at 12 hours after the reperfusion, administration of candesartan suppressed these expressions. The production of ROS that was detected at 12 hours after reperfusion was also suppressed by the administration of candesartan or apocynin. NADPH oxidase-mediated ROS production increased at 12 hours after reperfusion. Candesartan may protect neurons by decreasing extracellular glutamate immediately after reperfusion and by attenuating oxidative stress via a modulation of the AT1-R signaling that occurs during ischemic insult.

Co-administration of ethanol and nicotine: the enduring alterations in the rewarding properties of nicotine and glutamate activity within the mesocorticolimbic system of female alcohol-preferring (P) rats.

PubMed

Deehan, Gerald A; Hauser, Sheketha R; Waeiss, R Aaron; Knight, Christopher P; Toalston, Jamie E; Truitt, William A; McBride, William J; Rodd, Zachary A

2015-12-01

The co-abuse of ethanol (EtOH) and nicotine (NIC) increases the likelihood that an individual will relapse to drug use while attempting to maintain abstinence. There is limited research examining the consequences of long-term EtOH and NIC co-abuse. The current experiments determined the enduring effects of chronic EtOH, NIC, or EtOH + NIC intake on the reinforcing properties of NIC and glutamate (GLU) activity within the mesocorticolimbic (MCL) system. Alcohol-preferring (P) rats self-administered EtOH, Sacc + NIC, or EtOH + NIC combined for 10 weeks. The reinforcing properties of 0.1-3.0 μM NIC within the nucleus accumbens shell (AcbSh) were assessed following a 2-3-week drug-free period using intracranial self-administration (ICSA) procedures. The effects of EtOH, Sacc, Sacc + NIC, or EtOH + NIC intake on extracellular levels and clearance of glutamate (GLU) in the medial prefrontal cortex (mPFC) were also determined. Binge intake of EtOH (96-100 mg%) and NIC (21-27 mg/mL) were attained. All groups of P rats self-infused 3.0 μM NIC directly into the AcbSh, whereas only animals in the EtOH + NIC co-abuse group self-infused the 0.3 and 1.0 μM NIC concentrations. Additionally, self-administration of EtOH + NIC, but not EtOH, Sacc or Sacc + NIC, resulted in enduring increases in basal extracellular GLU levels in the mPFC. Overall, the co-abuse of EtOH + NIC produced enduring neuronal alterations within the MCL which enhanced the rewarding properties of NIC in the AcbSh and elevated extracellular GLU levels within the mPFC.

Proof of mechanism study of a novel serotonin transporter blocker, DA-8031, using [11C]DASB positron emission tomography and in vivo microdialysis.

PubMed

Park, Hyun Soo; Jung, In Soon; Lim, Nam Hee; Sung, Ji Hyun; Lee, Sukhyang; Moon, Byung Seok; Lee, Byung Chul; Kang, Kyung Koo; Kim, Sang Eun

2014-07-01

To investigate the efficacy of DA-8031, a novel compound for the treatment of premature ejaculation, we measured serotonin transporter (SERT) occupancy by DA-8031, as well as DA-8031-induced changes in extracellular serotonin levels, in the rat brain using positron emission tomography (PET) and 11C-N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine ([11C]DASB) and in vivo microdialysis, respectively. [11C]DASB PET scans were performed in rats with graded doses of DA-8031 (vehicle: 10, 30, and 100 mg/kg). SERT occupancy in the midbrain was determined using binding potentials for [11C]DASB calculated by the multilinear reference tissue model. Extracellular serotonin levels were monitored in the dorsal raphe nucleus of rats after the administration of DA-8031 (10-100 mg/kg) using in vivo microdialysis. PET data indicated a reduction of [11C]DASB binding to SERTs in the midbrain as a function of DA-8031 dose. SERT occupancy for each DA-8031 dose (10-100 mg/kg) ranged between 31% and 84%. The drug dose required for 50% occupancy of SERT was 13.5 mg/kg in the midbrain, comparable with previous preclinical behavioral data (∼10-30 mg/kg). In vivo microdialysis showed that DA-8031 produced a dose-dependent increase in extracellular serotonin levels in the dorsal raphe nucleus (33%-81% increase for doses of 10-100 mg/kg). These preclinical data provide a proof of mechanism for DA-8031 as a novel compound of targeting the SERT for the treatment of premature ejaculation, warranting further clinical trials. They also offer insight into the optimal drug dose needed to exert therapeutic effects while minimizing adverse effects in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing*

PubMed Central

Saha, Kaustuv; Sambo, Danielle; Richardson, Ben D.; Lin, Landon M.; Butler, Brittany; Villarroel, Laura; Khoshbouei, Habibeh

2014-01-01

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na+ or Cl− ion. Although isosmotic substitution of extracellular Na+ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl− ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons. PMID:24962577

Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness.

PubMed

Saladi, Srinivas Vinod; Keenen, Bridget; Marathe, Himangi G; Qi, Huiling; Chin, Khew-Voon; de la Serna, Ivana L

2010-10-22

Metastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro. To characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene. We provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

PubMed

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Therapeutic doses of oral methylphenidate significantly increase extracellular dopamine in the human brain.

PubMed

Volkow, N D; Wang, G; Fowler, J S; Logan, J; Gerasimov, M; Maynard, L; Ding, Y; Gatley, S J; Gifford, A; Franceschi, D

2001-01-15

Methylphenidate (Ritalin) is the most commonly prescribed psychoactive drug in children for the treatment of attention deficit hyperactivity disorder (ADHD), yet the mechanisms responsible for its therapeutic effects are poorly understood. Whereas methylphenidate blocks the dopamine transporter (main mechanism for removal of extracellular dopamine), it is unclear whether at doses used therapeutically it significantly changes extracellular dopamine (DA) concentration. Here we used positron emission tomography and [(11)C]raclopride (D2 receptor radioligand that competes with endogenous DA for binding to the receptor) to evaluate whether oral methylphenidate changes extracellular DA in the human brain in 11 healthy controls. We showed that oral methylphenidate (average dose 0.8 +/- 0.11 mg/kg) significantly increased extracellular DA in brain, as evidenced by a significant reduction in B(max)/K(d) (measure of D2 receptor availability) in striatum (20 +/- 12%; p < 0.0005). These results provide direct evidence that oral methylphenidate at doses within the therapeutic range significantly increases extracellular DA in human brain. This result coupled with recent findings of increased dopamine transporters in ADHD patients (which is expected to result in reductions in extracellular DA) provides a mechanistic framework for the therapeutic efficacy of methylphenidate. The increase in DA caused by the blockade of dopamine transporters by methylphenidate predominantly reflects an amplification of spontaneously released DA, which in turn is responsive to environmental stimulation. Because DA decreases background firing rates and increases signal-to-noise in target neurons, we postulate that the amplification of weak DA signals in subjects with ADHD by methylphenidate would enhance task-specific signaling, improving attention and decreasing distractibility. Alternatively methylphenidate-induced increases in DA, a neurotransmitter involved with motivation and reward, could enhance the salience of the task facilitating the "interest that it elicits" and thus improving performance.

Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice.

PubMed

Altamirano, Francisco; Valladares, Denisse; Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R; Allen, Paul D; Jaimovich, Enrique

2013-01-01

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca(2+)]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+)]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+)]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+)]r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.

Nifedipine Treatment Reduces Resting Calcium Concentration, Oxidative and Apoptotic Gene Expression, and Improves Muscle Function in Dystrophic mdx Mice

PubMed Central

Henríquez-Olguín, Carlos; Casas, Mariana; López, Jose R.; Allen, Paul D.; Jaimovich, Enrique

2013-01-01

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca2+]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca2+]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca2+]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91phox/p47phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca2+]r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD. PMID:24349043

Influence of sildenafil and donepezil administration on the serum redox balance in experimentally induced lower limb critical ischemia.

PubMed

Constantinescu, Mihaela Ioana; Constantinescu, Dan Petru; Andercou, Aurel; Mironiuc, Ion Aurel

2013-01-01

Chronic lower limb ischemia (CLLI) leads to endothelial cell dysfunctions and endothelial lesions. The use of substances that release nitric oxide and activate endothelial nitric oxide synthase has proved to be useful in increasing angiogenesis and arteriogenesis under critical ischemia conditions. To investigate the therapeutic effect of Sildenafil and Donepezil with a vasodilating action in experimentally induced CLLI and on serum redox homeostasis. The research was performed in 3 groups of rats (n=10 animals/group) with experimentally induced CLLI: group I - control group; group II - animals treated postoperatively with a therapeutic dose of sildenafil, and group III - animals treated postoperatively with a therapeutic dose of donepezil. Oxidative stress (OS) indicators (malondialdehyde - MDA, protein carbonyls - PC), antioxidant (AO) defense indicators (reduced glutathione - GSH and oxidized glutathione - GSSH), and ceruloplasmin (CP) were determined on days 7, 14, 21 and 30. Statistical processing was performed using the Excel application (Microsoft Office 2007), with the StatsDirect v.2.7.2 software. Changes in OS were evidenced in all groups on account of a decrease in MDA and PC. The greatest OS decrease in all groups was on day 30. AO defence changes were represented by decreased levels of GSH and GSSG in all groups, at the studied moments. Intracellular AO defense in the cytosol, nucleus and mitochondria was similar in all groups, (decreased GSH, GSSG and GSH/GSSG ratio). We found increased extracellular levels of GSH, GSSG, and CP and increased extracellular GSH/GSSG ratio at level compared to values on day 7. 1) The administration of sildenafil (group II) and donepezil (group III) has favorable effects on reducing OS in experimentally induced CLLI. 2) Sildenafil and Donepezil administration stimulates extracellular AO defense on account of CP. 3) Sildenafil and Donepezil administration influences intracellular redox homeostasis on account of the GSH/GSSG couple, the major redox buffer in the body.

Metabolic and histologic effects of sodium pyruvate treatment in the rat after cortical contusion injury.

PubMed

Fukushima, Masamichi; Lee, Stefan M; Moro, Nobuhiro; Hovda, David A; Sutton, Richard L

2009-07-01

This study determined the effects of intraperitoneal sodium pyruvate (SP) treatment on the levels of circulating fuels and on cerebral microdialysis levels of glucose (MD(glc)), lactate (MD(lac)), and pyruvate (MD(pyr)), and the effects of SP treatment on neuropathology after left cortical contusion injury (CCI) in rats. SP injection (1000 mg/kg) 5 min after sham injury (Sham-SP) or CCI (CCI-SP) significantly increased arterial pyruvate (p < 0.005) and lactate (p < 0.001) compared to that of saline-treated rats with CCI (CCI-Sal). Serum glucose also increased significantly in CCI-SP compared to that in CCI-Sal rats (p < 0.05), but not in Sham-SP rats. MD(pyr) was not altered after CCI-Sal, whereas MD(lac) levels within the cerebral cortex significantly increased bilaterally (p < 0.05) and those for MD(glc) decreased bilaterally (p < 0.05). MD(pyr) levels increased significantly in both Sham-SP and CCI-SP rats (p < 0.05 vs. CCI-Sal) and were higher in left/injured cortex of the CCI-SP group (p < 0.05 vs. sham-SP). In CCI-SP rats the contralateral MD(lac) decreased below CCI-Sal levels (p < 0.05) and the ipsilateral MD(glc) levels exceeded those of CCI-Sal rats (p < 0.05). Rats with a single low (500 mg/kg) or high dose (1000 mg/kg) SP treatment had fewer damaged cortical cells 6 h post-CCI than did saline-treated rats (p < 0.05), but three hourly injections of SP (1000 mg/kg) were needed to significantly reduce contusion volume 2 weeks after CCI. Thus, a single intraperitoneal SP treatment increases circulating levels of three potential brain fuels, attenuates a CCI-induced reduction in extracellular glucose while increasing extracellular levels of pyruvate, but not lactate, and can attenuate cortical cell damage occurring within 6 h of injury. Enduring (2 week) neuronal protection was achieved only with multiple SP treatments within the first 2 h post-CCI, perhaps reflecting the need for additional fuel throughout the acute period of increased metabolic demands induced by CCI.

Metabolic and Histologic Effects of Sodium Pyruvate Treatment in the Rat after Cortical Contusion Injury

PubMed Central

Fukushima, Masamichi; Lee, Stefan M.; Moro, Nobuhiro; Hovda, David A.

2009-01-01

Abstract This study determined the effects of intraperitoneal sodium pyruvate (SP) treatment on the levels of circulating fuels and on cerebral microdialysis levels of glucose (MDglc), lactate (MDlac), and pyruvate (MDpyr), and the effects of SP treatment on neuropathology after left cortical contusion injury (CCI) in rats. SP injection (1000 mg/kg) 5 min after sham injury (Sham-SP) or CCI (CCI-SP) significantly increased arterial pyruvate (p < 0.005) and lactate (p < 0.001) compared to that of saline-treated rats with CCI (CCI-Sal). Serum glucose also increased significantly in CCI-SP compared to that in CCI-Sal rats (p < 0.05), but not in Sham-SP rats. MDpyr was not altered after CCI-Sal, whereas MDlac levels within the cerebral cortex significantly increased bilaterally (p < 0.05) and those for MDglc decreased bilaterally (p < 0.05). MDpyr levels increased significantly in both Sham-SP and CCI-SP rats (p < 0.05 vs. CCI-Sal) and were higher in left/injured cortex of the CCI-SP group (p < 0.05 vs. sham-SP). In CCI-SP rats the contralateral MDlac decreased below CCI-Sal levels (p < 0.05) and the ipsilateral MDglc levels exceeded those of CCI-Sal rats (p < 0.05). Rats with a single low (500 mg/kg) or high dose (1000 mg/kg) SP treatment had fewer damaged cortical cells 6 h post-CCI than did saline-treated rats (p < 0.05), but three hourly injections of SP (1000 mg/kg) were needed to significantly reduce contusion volume 2 weeks after CCI. Thus, a single intraperitoneal SP treatment increases circulating levels of three potential brain fuels, attenuates a CCI-induced reduction in extracellular glucose while increasing extracellular levels of pyruvate, but not lactate, and can attenuate cortical cell damage occurring within 6 h of injury. Enduring (2 week) neuronal protection was achieved only with multiple SP treatments within the first 2 h post-CCI, perhaps reflecting the need for additional fuel throughout the acute period of increased metabolic demands induced by CCI. PMID:19594384

Synergistic role of HSP90α and HSP90β to promote myofibroblast persistence in lung fibrosis.

PubMed

Bellaye, Pierre-Simon; Shimbori, Chiko; Yanagihara, Toyoshi; Carlson, David A; Hughes, Philip; Upagupta, Chandak; Sato, Seidai; Wheildon, Nolan; Haystead, Timothy; Ask, Kjetil; Kolb, Martin

2018-02-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90β, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90β was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-β1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90β stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF. Copyright ©ERS 2018.

Mast cells exert pro-inflammatory effects of relevance to the pathophyisology of tendinopathy.

PubMed

Behzad, Hayedeh; Sharma, Aishwariya; Mousavizadeh, Rouhollah; Lu, Alex; Scott, Alex

2013-01-01

We have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls. This study examined the influence of mast cells on basic tenocyte functions, including production of the inflammatory mediator prostaglandin E2 (PGE2), extracellular matrix remodeling and matrix metalloproteinase (MMP) gene transcription, and collagen synthesis. Primary human tenocytes were stimulated with an established human mast cell line (HMC-1). Extracellular matrix remodeling was studied by culturing tenocytes in a three-dimensional collagen lattice. Survival/proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Levels of mRNA for COX-2, COL1A1, MMP1, and MMP7 were determined by quantitative real-time polymerase chain reaction (qPCR). Cox-2 protein level was assessed by Western blot analysis and type I procollagen was detected by immunofluorescent staining. PGE2 levels were determined using an enzyme-linked immunosorbent assay (ELISA). Mast cells stimulated tenocytes to produce increased levels of COX-2 and the pro-inflammatory mediator PGE2, which in turn decreased COL1A1 mRNA expression. Additionally, mast cells reduced the type I procollagen protein levels produced by tenocytes. Transforming growth factor beta 1 (TGF-β1) was responsible for the induction of Cox-2 and PGE2 by tenocytes. Mast cells increased MMP1 and MMP7 transcription and increased the contraction of a three-dimensional collagen lattice by tenocytes, a phenomenon which was blocked by a pan-MMP inhibitor (Batimastat). Our data demonstrate that mast cell-derived PGE2 reduces collagen synthesis and enhances expression and activities of MMPs in human tenocytes.

Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate12

PubMed Central

Feng, Lili; Sun, Xiaofeng; Csizmadia, Eva; Han, Lihui; Bian, Shu; Murakami, Takashi; Wang, Xin; Robson, Simon C; Wu, Yan

2011-01-01

Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5′-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X7 receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X7 expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management. PMID:21390184

Activation of Extracellular Signal-Regulated Kinases (ERK 1/2) in the Locus Coeruleus Contributes to Pain-Related Anxiety in Arthritic Male Rats

PubMed Central

Borges, Gisela; Miguelez, Cristina; Neto, Fani; Mico, Juan Antonio; Ugedo, Luisa

2017-01-01

Abstract Background: There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety. Methods: Using the complete Freund’s adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction. Results: The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered. Conclusion: As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety. PMID:28158734

Activation of Extracellular Signal-Regulated Kinases (ERK 1/2) in the Locus Coeruleus Contributes to Pain-Related Anxiety in Arthritic Male Rats.

PubMed

Borges, Gisela; Miguelez, Cristina; Neto, Fani; Mico, Juan Antonio; Ugedo, Luisa; Berrocoso, Esther

2017-06-01

There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety. Using the complete Freund's adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction. The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered. As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety. © The Author 2017. Published by Oxford University Press on behalf of CINP.

Thiopental sodium reduces glutamate extracellular levels in rat intact prefrontal cortex.

PubMed

Liu, Hongliang; Yao, Shanglong

2005-12-01

To investigate the effect of thiopental sodium on glutamate extracellular levels in the prefrontal cortex (PFC) of rats, a microdialysis probe was inserted into the PFC, the perfusate was collected every 10 min throughout the experiment with thiopental sodium ip or perfused into the PFC locally. The concentrations of glutamate in the perfusate were determined by reversed-phase high performance liquid chromatography. Thiopental sodium 30 mg kg(-1) ip significantly decreased glutamate levels in the perfusate after 10, 20, 30, and 40 min; glutamate levels in the perfusate were also decreased from 10 to 90 min after thiopental sodium 50 mg kg(-1) ip. Thiopental sodium with concentrations of 30, 100, or 300 microM perfused into the PFC also decreased glutamate levels in the perfusate significantly. The results suggest that thiopental sodium decreases glutamate extracellular levels in rat intact PFC.

Hypoxia Induces an Increase in Intracellular Magnesium via Transient Receptor Potential Melastatin 7 (TRPM7) Channels in Rat Hippocampal Neurons in Vitro*

PubMed Central

Zhang, Jing; Zhao, Fengbo; Zhao, Yin; Wang, Jing; Pei, Lei; Sun, Ning; Shi, Jing

2011-01-01

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg2+ fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg2+]i) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg2+]o). The results showed that the neuronal [Mg2+]i was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg2+]i reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg2+]o decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg2+]i peak immediately appeared in hippocampal neurons. This increase of [Mg2+]i declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd3+ or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg2+]i increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg2+]i. By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg2+]i induced by OGD or CI but also the basal levels of [Mg2+]i were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg2+]i after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg2+]i via TRPM7 channels in rat hippocampal neurons. PMID:21487014

Sleep/wake dependent changes in cortical glucose concentrations.

PubMed

Dash, Michael B; Bellesi, Michele; Tononi, Giulio; Cirelli, Chiara

2013-01-01

Most of the energy in the brain comes from glucose and supports glutamatergic activity. The firing rate of cortical glutamatergic neurons, as well as cortical extracellular glutamate levels, increase with time spent awake and decline throughout non rapid eye movement sleep, raising the question whether glucose levels reflect behavioral state and sleep/wake history. Here chronic (2-3 days) electroencephalographic recordings in the rat cerebral cortex were coupled with fixed-potential amperometry to monitor the extracellular concentration of glucose ([gluc]) on a second-by-second basis across the spontaneous sleep-wake cycle and in response to 3 h of sleep deprivation. [Gluc] progressively increased during non rapid eye movement sleep and declined during rapid eye movement sleep, while during wake an early decline in [gluc] was followed by an increase 8-15 min after awakening. There was a significant time of day effect during the dark phase, when rats are mostly awake, with [gluc] being significantly lower during the last 3-4 h of the night relative to the first 3-4 h. Moreover, the duration of the early phase of [gluc] decline during wake was longer after prolonged wake than after consolidated sleep. Thus, the sleep/wake history may affect the levels of glucose available to the brain upon awakening. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Pyruvate and oxaloacetate limit zinc-induced oxidative HT-22 neuronal cell injury.

PubMed

Berry, Elizabeth V; Toms, Nick J

2006-12-01

During CNS ischaemia, accumulating evidence suggests that raised intracellular Zn(2+) levels may play a significant role in inducing neuronal cell death. Several mechanisms mediating Zn(2+)-induced cell death have been suggested, however the precise molecular mechanisms remain uncertain. Employing the HT-22 murine hippocampal neuronal cell line, we have evaluated possible mechanisms of cytotoxic extracellular Zn(2+) insults. Increased extracellular Zn(2+) levels was found to induce concentration-dependent cytotoxicity. When tested at 200muM, Zn(2+) increased intracellular Zn(2+) levels (determined via FluoZin-3 fluorescence) and rapidly induced cell death. However, neither L-type (nimodipine) nor T-type (mibefradil) voltage-activated Ca(2+) channel inhibitors limited Zn(2+)-induced cytotoxicity. Furthermore, and in contrast with staurosporine, Zn(2+) cytotoxic insults failed to induce significant caspase-3 activation and were insensitive to the poly-caspase inhibitor, zVAD-fmk. Antioxidant co-application (Trolox and N,N'-diphenyl-1,4-phenylenediamine (DPPD)) was neuroprotective versus 6h Zn(2+) insults. Additionally, despite inducing significant mitochondrial membrane potential loss, Zn(2+) failed to induce detectable increased superoxide production. However, both pyruvate and oxaloacetate were found to afford significant neuroprotection versus Zn(2+) cytotoxic insults, without significantly influencing intracellular Zn(2+) accumulation. We conclude that cultured HT-22 neurones are vulnerable to Zn(2+) cytotoxic insults via a non-caspase-3 mediated mechanism, which involves glycolytic inhibition.

Differential release and deposition of S100A8/A9 proteins in inflamed upper airway tissue.

PubMed

Van Crombruggen, Koen; Vogl, Thomas; Pérez-Novo, Claudina; Holtappels, Gabriele; Bachert, Claus

2016-01-01

Intracellular Ca(2+)-binding S100A8/A9 proteins gain novel functions when released during inflammation. The exact outcome of their extracellular function depends on the local tissue environment in which they are released; both anti-inflammatory and pro-inflammatory responses are described, modulating the immune system by binding Toll-like receptor (TLR)-4 or the receptor for advanced glycation end-products (RAGE). However, the contribution of the proteins in the pathophysiology of chronic rhinosinusitis (CRS) remains unclear.Homomeric S100A8 and S100A9, and heteromeric S100A8/A9 proteins were evaluated in CRS with/without nasal polyps (CRSw/sNP) and controls. Functional responses were assessed in polyp tissue stimulated with S100 proteins in the presence of TLR-4 and RAGE blocking antibodies.S100A8, S100A9 and S100A8/A9 protein levels were significantly higher in CRSwNP patients, showing increased deposition on extracellular matrix (ECM) structures of CRSwNP tissue in contrast to CRSsNP and controls. In the presence of Staphylococcus aureus, S100A8/A9 is released from neutrophils and from the ECM. Extracellular S100A8 and S100A9 proteins induced increased levels of diverse inflammatory mediators via TLR-4 engagement.The inflammatory/remodelling characteristics of CRSwNP specifically allow increased retention of S100A8, S100A9 and S100A8/A9 proteins in the ECM of CRSwNP tissue. Upon release, homodimeric proteins act as a local danger signal inducing inflammatory mediators, predominantly via TLR-4 activation. Copyright ©ERS 2016.

Cardiac basal metabolism: energetic cost of calcium withdrawal in the adult rat heart.

PubMed

Bonazzola, P; Takara, D

2010-07-01

Cardiac basal metabolism upon extracellular calcium removal and its relationship with intracellular sodium and calcium homeostasis was evaluated. A mechano-calorimetric technique was used that allowed the simultaneous and continuous measurement of both heat rate and resting pressure in arterially perfused quiescent adult rat hearts. Using pharmacological tools, the possible underlying mechanisms related to sodium and calcium movements were investigated. Resting heat rate (expressed in mW g(-1)(dry wt)) increased upon calcium withdrawal (+4.4 +/- 0.2). This response was: (1) unaffected by the presence of tetrodotoxin (+4.3 +/- 0.6), (2) fully blocked by both, the decrease in extracellular sodium concentration and the increase in extracellular magnesium concentration, (3) partially blocked by the presence of either nifedipine (+2.8 +/- 0.4), KB-R7943 (KBR; +2.5 +/- 0.2), clonazepam (CLO; +3.1 +/- 0.3) or EGTA (+1.9 +/- 0.3). The steady heat rate under Ca(2+)-free conditions was partially reduced by the addition of Ru360 (-1.1 +/- 0.2) but not CLO in the presence of EGTA, KBR or Ru360. Energy expenditure for resting state maintenance upon calcium withdrawal depends on the intracellular rise in both sodium and calcium. Our data are consistent with a mitochondrial Ca(2+) cycling, not detectable under normal calcium diastolic levels. The experimental condition here analysed, partially simulates findings reported under certain pathological situations including heart failure in which mildly increased levels of both diastolic sodium and calcium have also been found. Therefore, under such pathological conditions, hearts should distract chemical energy to fuel processes associated with sodium and calcium handling, making more expensive the maintenance of their functions.

Influence of statins on distinct circulating microRNAs during prolonged aerobic exercise

PubMed Central

Min, Pil-Ki; Park, Joseph; Isaacs, Stephanie; Taylor, Beth A.; Thompson, Paul D.; Troyanos, Chris; D'Hemecourt, Pierre; Dyer, Sophia; Baggish, Aaron L.

2015-01-01

Statins exacerbate exercise-induced skeletal muscle injury. Muscle-specific microRNAs (myomiRs) increase in plasma after prolonged exercise, but the patterns of myomiRs release after statin-associated muscle injury have not been examined. We examined the relationships between statin exposure, in vitro and in vivo muscle contraction, and expression of candidate circulating myomiRs. We measured plasma levels of myomiRs, circulating microRNA-1 (c-miR-1), c-miR-133a, c-miR-206, and c-miR-499-5p from 28 statin-using and 28 nonstatin-using runners before (PRE), immediately after (FINISH), and 24 h after they ran a 42-km footrace (the 2011 Boston marathon) (POST-24). To examine these cellular-regulation myomiRs, we used contracting mouse C2C12 myotubes in culture with and without statin exposure to compare intracellular and extracellular expression of these molecules. In marathoners, c-miR-1, c-miR-133a, and c-miR-206 increased at FINISH, returned to baseline at POST-24, and were unaffected by statin use. In contrast, c-miR-499-5p was unchanged at FINISH but increased at POST-24 among statin users compared with PRE and runners who did not take statins. In cultured C2C12 myotubes, extracellular c-miR-1, c-miR-133a, and c-miR-206 were significantly increased by muscle contraction regardless of statin use. In contrast, extracellular miR-499-5p was unaffected by either isolated statin exposure or isolated carbachol exposure but it was increased when muscle contraction was combined with statin exposure. In summary, we found that statin-potentiated muscle injury during exercise is accompanied by augmented extracellular release of miR-499-5p. Thus c-miR-499-5p may serve as a biomarker of statin-potentiated muscle damage. PMID:26472872

Reduced prefrontal dopaminergic activity in valproic acid-treated mouse autism model.

PubMed

Hara, Yuta; Takuma, Kazuhiro; Takano, Erika; Katashiba, Keisuke; Taruta, Atsuki; Higashino, Kosuke; Hashimoto, Hitoshi; Ago, Yukio; Matsuda, Toshio

2015-08-01

Previous studies suggest that dysfunction of neurotransmitter systems is associated with the pathology of autism in humans and the disease model rodents, but the precise mechanism is not known. Rodent offspring exposed prenatally to VPA shows autism-related behavioral abnormalities. The present study examined the effect of prenatal VPA exposure on brain monoamine neurotransmitter systems in male and female mice. The prenatal VPA exposure did not affect the levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT) and their metabolites in the prefrontal cortex and striatum, while it significantly reduced methamphetamine (METH) (1.0 mg/kg)-induced hyperlocomotion in male offspring. In vivo microdialysis study demonstrated that prenatal VPA exposure attenuated METH-induced increases in extracellular DA levels in the prefrontal cortex, while it did not affect those in extracellular NA and 5-HT levels. Prenatal VPA exposure also decreased METH-induced c-Fos expression in the prefrontal cortex and the mRNA levels of DA D1 and D2 receptors in the prefrontal cortex. These effects of VPA were not observed in the striatum. In contrast to male offspring, prenatal VPA exposure did not affect METH-induced increases in locomotor activity and prefrontal DA levels and the D1 and D2 receptor mRNA levels in the prefrontal cortex in female offspring. These findings suggest that prenatal VPA exposure causes hypofunction of prefrontal DA system in a sex-dependent way. Copyright © 2015 Elsevier B.V. All rights reserved.

High levels of the extracellular matrix proteoglycan decorin are associated with inhibition of testicular function

PubMed Central

Adam, Marion; Urbanski, Henryk F.; Garyfallou, Vasilios T.; Welsch, Ulrich; Köhn, Frank M.; Schwarzer, J. Ullrich; Strauss, Leena; Poutanen, Matti; Mayerhofer, Artur

2011-01-01

Decorin (DCN), a component of the extracellular matrix of the peritubular wall and the interstitial areas of the human testis, can interact with growth factor (GF) signaling, thereby blocking downstream actions of GFs. In the present study the expression and regulation of DCN using both human testes and two experimental animal models, namely the rhesus monkey and mouse, were examined. DCN protein was present in peritubular and interstitial areas of adult human and monkey testes, while it was almost undetectable in adult wild-type mice. Interestingly, the levels and sites of testicular DCN expression in the monkeys were inversely correlated with testicular maturation markers. A strong DCN expression associated with the abundant connective tissue of the interstitial areas in the postnatal through prepubertal phases was observed. In adult and old monkeys the DCN pattern was similar to the one in normal human testes, presenting strong expression at the peritubular region. In the testes of both infertile men and in a mouse model of inflammation associated infertility (aromatase-overexpressing transgenic mice), the fibrotic changes and increased numbers of Tumor necrosis factor (TNF)-α-producing immune cells were shown to be associated with increased production of DCN. Furthermore, studies with human testicular peritubular cells isolated from fibrotic testis indicated that TNF-α significantly increased DCN production. The data, thus, show that an increased DCN level is associated with impaired testicular function, supporting our hypothesis that DCN interferes with paracrine signaling of the testis in health and disease. PMID:22413766

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

PubMed

Wang, Shenqiu; Tsarouhas, Vasilios; Xylourgidis, Nikos; Sabri, Nafiseh; Tiklová, Katarína; Nautiyal, Naumi; Gallio, Marco; Samakovlis, Christos

2009-07-01

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium.

PubMed

Shynlova, Oksana; Mitchell, Jennifer A; Tsampalieros, Anne; Langille, B Lowell; Lye, Stephen J

2004-04-01

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.

Conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: Acid-base status, trace elements and δ11B

NASA Astrophysics Data System (ADS)

Heinemann, Agnes; Fietzke, Jan; Melzner, Frank; BöHm, Florian; Thomsen, JöRn; Garbe-SchöNberg, Dieter; Eisenhauer, Anton

2012-01-01

Mytilus edulis were cultured for 3 months under six different seawater pCO2 levels ranging from 380 to 4000 μatm. Specimen were taken from Kiel Fjord (Western Baltic Sea, Germany) which is a habitat with high and variable seawater pCO2 and related shifts in carbonate system speciation (e.g., low pH and low CaCO3 saturation state). Hemolymph (HL) and extrapallial fluid (EPF) samples were analyzed for pH and total dissolved inorganic carbon (CT) to calculate pCO2 and [HCO3-]. A second experiment was conducted for 2 months with three different pCO2 levels (380, 1400 and 4000 μatm). Boron isotopes (δ11B) were investigated by LA-MC-ICP-MS (Laser Ablation-Multicollector-Inductively Coupled Plasma-Mass Spectrometry) in shell portions precipitated during experimental treatment time. Additionally, elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF of specimen from the second experiment were measured via ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Extracellular pH was not significantly different in HL and EPF but systematically lower than ambient water pH. This is due to high extracellular pCO2 values, a prerequisite for metabolic CO2 excretion. No accumulation of extracellular [HCO3-] was measured. Elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF increased slightly with pH which is in accordance with increasing growth and calcification rates at higher seawater pH values. Boron isotope ratios were highly variable between different individuals but also within single shells. This corresponds to a high individual variability in fluid B/Ca ratios and may be due to high boron concentrations in the organic parts of the shell. The mean δ11B value shows no trend with pH but appears to represent internal pH (EPF) rather than ambient water pH.

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

PubMed

Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

2000-02-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

PubMed Central

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

Volume regulation in mammalian skeletal muscle: the role of sodium-potassium-chloride cotransporters during exposure to hypertonic solutions.

PubMed

Lindinger, Michael I; Leung, Matthew; Trajcevski, Karin E; Hawke, Thomas J

2011-06-01

Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the degree of change in extracellular osmolarity, is muscle specific, and is dependent on the functioning of the NKCC and Na+, K+-ATPase.

Sirt1 S-nitrosylation induces acetylation of HMGB1 in LPS-activated RAW264.7 cells and endotoxemic mice.

PubMed

Kim, Young Min; Park, Eun Jung; Kim, Hye Jung; Chang, Ki Churl

2018-06-18

Excessive inflammation plays a detrimental role in endotoxemia. A recent study indicated that alarmins such as high mobility group box 1 (HMGB1) have drawn attention as therapeutic targets of sepsis. Post-translational modification (i.e., acetylation of lysine residues) of HMGB1 leads to the release of HMGB1 into the cellular space, operating as a warning signal that induces inflammation. Sirtuin 1 (SIRT1) has been shown to negatively regulate HMGB1 hyperacetylation and its extracellular release in sepsis. Therefore, we hypothesized that the S-nitrosylation (SNO) of SIRT1 may disrupt the ability of SIRT1 to negatively regulate the hyperacetylation of HMGB1. As long as the S-nitrosylation of SIRT1 occurs during septic conditions, it may worsen the situation. We found that the activity of SIRT1 decreased as the SNO-SIRT1 levels increased, resulting in HMGB1 release by LPS in RAW264.7 cells. Both the iNOS inhibitor (1400 W) and silencing iNOS significantly inhibited SNO-SIRT1, allowing increases in SIRT1 activity that decreased the HMGB1 release by LPS. SNAP, a NO donor, significantly increased both SNO-SIRT1 levels and the HMGB1 release that was accompanied by decreased sirt1 activity. However, sirtinol, a Sirt1 inhibitor, by itself decreased Sirt1 activity compared to that of the control, so that it did not affect already increased SNO-SIRT levels by SNAP. Most importantly, in lung tissues of LPS-endotoxic mice, significantly increased levels of SNO-SIRT were found, which was inhibited by 1400 W treatment. Plasma nitrite and HMGB1 levels were significantly higher than those in the sham controls, and the elevated levels were significantly lowered in the presence of 1400 W. We concluded that the S-nitrosylation of Sirt1 under endotoxic conditions may uninhibit the acetylation of HMGB1 and its extracellular release. Copyright © 2018 Elsevier Inc. All rights reserved.

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium*

PubMed Central

Aihara, Eitaro; Hentz, Courtney L.; Korman, Abraham M.; Perry, Nicholas P. J.; Prasad, Vikram; Shull, Gary E.; Montrose, Marshall H.

2013-01-01

We report that a localized intracellular and extracellular Ca2+ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca2+-sensitive protein (yellow cameleon 3.0) report that intracellular Ca2+ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca2+ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca2+ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca2+ mobilization. Indomethacin and verapamil also inhibit the luminal Ca2+ increase. Intracellular Ca2+ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca2+ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca2+ and unevenly inhibits late-phase intracellular Ca2+ mobilization. Both modes of Ca2+ chelation slow gastric repair. In plasma membrane Ca-ATPase 1+/− mice, but not plasma membrane Ca-ATPase 4−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca2+ increase. We conclude that endogenous Ca2+, mobilized by signaling pathways and transmembrane Ca2+ transport, causes increased Ca2+ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo. PMID:24121509

In vivo epithelial wound repair requires mobilization of endogenous intracellular and extracellular calcium.

PubMed

Aihara, Eitaro; Hentz, Courtney L; Korman, Abraham M; Perry, Nicholas P J; Prasad, Vikram; Shull, Gary E; Montrose, Marshall H

2013-11-22

We report that a localized intracellular and extracellular Ca(2+) mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca(2+)-sensitive protein (yellow cameleon 3.0) report that intracellular Ca(2+) selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca(2+) increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca(2+) increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca(2+) mobilization. Indomethacin and verapamil also inhibit the luminal Ca(2+) increase. Intracellular Ca(2+) chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca(2+) increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N'-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca(2+) and unevenly inhibits late-phase intracellular Ca(2+) mobilization. Both modes of Ca(2+) chelation slow gastric repair. In plasma membrane Ca-ATPase 1(+/-) mice, but not plasma membrane Ca-ATPase 4(-/-) mice, there is slowed epithelial repair and a diminished gastric surface Ca(2+) increase. We conclude that endogenous Ca(2+), mobilized by signaling pathways and transmembrane Ca(2+) transport, causes increased Ca(2+) levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.

KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

PubMed

Pampalakis, Georgios; Sykioti, Vasia-Samantha; Ximerakis, Methodios; Stefanakou-Kalakou, Ioanna; Melki, Ronald; Vekrellis, Kostas; Sotiropoulou, Georgia

2017-02-28

KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-to-cell propagation in "a prion-like mechanism". Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies.

KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species

PubMed Central

Pampalakis, Georgios; Sykioti, Vasia-Samantha; Ximerakis, Methodios; Stefanakou-Kalakou, Ioanna; Melki, Ronald; Vekrellis, Kostas; Sotiropoulou, Georgia

2017-01-01

KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-to-cell propagation in “a prion-like mechanism”. Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies. PMID:27845893

Serum levels of the extracellular domain of the epidermal growth factor receptor in individuals exposed to arsenic in drinking water in Bangladesh.

PubMed

Li, Y; Chen, Y; Slavkovic, V; Ahsan, H; Parvez, F; Graziano, J H; Brandt-Rauf, P W

2007-01-01

Epidermal growth factor receptor-dependent mechanisms have been implicated in growth signal transduction pathways that contribute to cancer development, including dermal carcinogenesis. Detection of the extracellular domain of the epidermal growth factor receptor (EGFR ECD) in serum has been suggested as a potential biomarker for monitoring this effect in vivo. Arsenic is a known human carcinogen, producing skin and other malignancies in populations exposed through their drinking water. One such exposed population, which we have been studying for a number of years, is in Bangladesh. The purpose of this study was to examine the EGFR ECD as a potential biomarker of arsenic exposure and/or effect in this population. Levels of the EGFR ECD were determined by enzyme-linked immunosorbent assay in the serum samples from 574 individuals with a range of arsenic exposures from drinking water in the Araihazar area of Bangladesh. In multiple regression analysis, serum EGFR ECD was found to be positively associated with three different measures of arsenic exposure (well water arsenic, urinary arsenic and a cumulative arsenic index) at statistically significant levels (p

In Vivo d-Serine Hetero-Exchange through Alanine-Serine-Cysteine (ASC) Transporters Detected by Microelectrode Biosensors

PubMed Central

2013-01-01

d-Serine, a co-agonist of N-methyl d-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. d-Serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular d-serine levels in vivo are still unclear. d-Serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of d-serine extracellular concentration in vivo. Alternatively, exocytotic d-serine release has also been proposed. In this study, the dynamics of d-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of d-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. d-Serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in d-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release d-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased d-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that d-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of d-serine extracellular concentration. PMID:23581544

Extracellular glucose can fuel metabolism in red blood cells from high glycemic Atlantic cod (Gadus morhua) but not low glycemic short-horned sculpin (Myoxocephalus scorpius).

PubMed

Driedzic, William R; Clow, Kathy A; Short, Connie E

2014-11-01

Energy metabolism was assessed in red blood cells (RBCs) from Atlantic cod and short-horned sculpin, two species that have markedly different levels of blood glucose. The objective was to determine whether the level of extracellular glucose has an impact on rates of glucose metabolism. The blood glucose level was 2.5 mmol l(-1) in Atlantic cod and 0.2 mmol l(-1) in short-horned sculpin, respectively. Oxygen consumption, lactate production and glucose utilization were measured in whole blood and related to grams of RBCs. Glucose utilization was assessed by measuring both glucose disappearance and the production of (3)H2O from [2-(3)H]-glucose. RBCs from both species have an aerobic-based metabolism. In Atlantic cod, extracellular glucose is sufficient to provide the sum of glucosyl equivalents to support both oxidative metabolism and lactate production. In contrast, extracellular glucose can account for only 10% of the metabolic rate in short-horned sculpin RBCs. In both species, about 70% of glucose enters the RBCs via facilitated transport. The difference in rates of extracellular glucose utilization is related to the extremely low levels of blood glucose in short-horned sculpin. In this species energy metabolism by RBCs must be supported by alternative fuels. © 2014. Published by The Company of Biologists Ltd.

Stabilization, Rolling, and Addition of Other Extracellular Matrix Proteins to Collagen Hydrogels Improve Regeneration in Chitosan Guides for Long Peripheral Nerve Gaps in Rats.

PubMed

Gonzalez-Perez, Francisco; Cobianchi, Stefano; Heimann, Claudia; Phillips, James B; Udina, Esther; Navarro, Xavier

2017-03-01

Autograft is still the gold standard technique for the repair of long peripheral nerve injuries. The addition of biologically active scaffolds into the lumen of conduits to mimic the endoneurium of peripheral nerves may increase the final outcome of artificial nerve devices. Furthermore, the control of the orientation of the collagen fibers may provide some longitudinal guidance architecture providing a higher level of mesoscale tissue structure. To evaluate the regenerative capabilities of chitosan conduits enriched with extracellular matrix-based scaffolds to bridge a critical gap of 15 mm in the rat sciatic nerve. The right sciatic nerve of female Wistar Hannover rats was repaired with chitosan tubes functionalized with extracellular matrix-based scaffolds fully hydrated or stabilized and rolled to bridge a 15 mm nerve gap. Recovery was evaluated by means of electrophysiology and algesimetry tests and histological analysis 4 months after injury. Stabilized constructs enhanced the success of regeneration compared with fully hydrated scaffolds. Moreover, fibronectin-enriched scaffolds increased muscle reinnervation and number of myelinated fibers compared with laminin-enriched constructs. A mixed combination of collagen and fibronectin may be a promising internal filler for neural conduits for the repair of peripheral nerve injuries, and their stabilization may increase the quality of regeneration over long gaps. Copyright © 2017 by the Congress of Neurological Surgeons

Increased Interleukin-32 Levels in Obesity Promote Adipose Tissue Inflammation and Extracellular Matrix Remodeling: Effect of Weight Loss.

PubMed

Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Valentí, Víctor; Moncada, Rafael; Landecho, Manuel F; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

2016-12-01

Interleukin (IL)-32 is a recently described cytokine involved in the regulation of inflammation. We aimed to explore whether IL-32 could function as an inflammatory and angiogenic factor in human obesity and obesity-associated type 2 diabetes. Samples obtained from 90 subjects were used in the study. Obese patients exhibited higher expression levels of IL-32 in visceral adipose tissue (AT) as well as in subcutaneous AT and peripheral blood mononuclear cells. IL32 was mainly expressed by stromovascular fraction cells, and its expression was significantly enhanced by inflammatory stimuli and hypoxia, whereas no changes were found after the incubation with anti-inflammatory cytokines. The addition of exogenous IL-32 induced the expression of inflammation and extracellular matrix-related genes in human adipocyte cultures, and IL32-silenced adipocytes showed a downregulation of inflammatory genes. Furthermore, adipocyte-conditioned media obtained from obese patients increased IL32 gene expression in human monocyte cultures, whereas the adipocyte-conditioned media from lean volunteers had no effect on IL32 mRNA levels. These findings provide evidence, for the first time, about the inflammatory and remodeling properties of IL-32 in AT, implicating this cytokine in obesity-associated comorbidities. © 2016 by the American Diabetes Association.

Significant levels of extracellular reactive oxygen species produced by brown rot basidiomycetes on cellulose

Treesearch

Roni Cohen; Kenneth A. Jensen; Carl J. Houtman; Kenneth E. Hammel

2002-01-01

It is often proposed that brown rot basidiomycetes use extracellular reactive oxygen species (ROS) to accomplish the initial depolymerization of cellulose in wood, but little evidence has been presented to show that the fungi produce these oxidants in physiologically relevant quantities. We used [14C]phenethyl polyacrylate as a radical trap to estimate extracellular...

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

DOE PAGES

Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G.; ...

2016-03-22

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits,more » the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe 2O 3 (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.« less

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G.

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits,more » the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe 2O 3 (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.« less

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

PubMed Central

Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

2015-01-01

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

High frequency stimulation abolishes thalamic network oscillations: an electrophysiological and computational analysis

NASA Astrophysics Data System (ADS)

Lee, Kendall H.; Hitti, Frederick L.; Chang, Su-Youne; Lee, Dongchul C.; Roberts, David W.; McIntyre, Cameron C.; Leiter, James C.

2011-08-01

Deep brain stimulation (DBS) of the thalamus has been demonstrated to be effective for the treatment of epilepsy. To investigate the mechanism of action of thalamic DBS, we examined the effects of high frequency stimulation (HFS) on spindle oscillations in thalamic brain slices from ferrets. We recorded intracellular and extracellular electrophysiological activity in the nucleus reticularis thalami (nRt) and in thalamocortical relay (TC) neurons in the lateral geniculate nucleus, stimulated the slice using a concentric bipolar electrode, and recorded the level of glutamate within the slice. HFS (100 Hz) of TC neurons generated excitatory post-synaptic potentials, increased the number of action potentials in both TC and nRt neurons, reduced the input resistance, increased the extracellular glutamate concentration, and abolished spindle wave oscillations. HFS of the nRt also suppressed spindle oscillations. In both locations, HFS was associated with significant and persistent elevation in extracellular glutamate levels and suppressed spindle oscillations for many seconds after the cessation of stimulation. We simulated HFS within a computational model of the thalamic network, and HFS also disrupted spindle wave activity, but the suppression of spindle activity was short-lived. Simulated HFS disrupted spindle activity for prolonged periods of time only after glutamate release and glutamate-mediated activation of a hyperpolarization-activated current (Ih) was incorporated into the model. Our results suggest that the mechanism of action of thalamic DBS as used in epilepsy may involve the prolonged release of glutamate, which in turn modulates specific ion channels such as Ih, decreases neuronal input resistance, and abolishes thalamic network oscillatory activity.

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Day, Danton H., E-mail: danton.oday@utoronto.ca; Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6; Huber, Robert J.

2012-09-07

Highlights: Black-Right-Pointing-Pointer Extracellular calmodulin is present throughout growth and development in Dictyostelium. Black-Right-Pointing-Pointer Extracellular calmodulin localizes within the ECM during development. Black-Right-Pointing-Pointer Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. Black-Right-Pointing-Pointer Extracellular calmodulin exists in eukaryotic microbes. Black-Right-Pointing-Pointer Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca{sup 2+}/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of themore » research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.« less

In vitro and in vivo physiology of low nanomolar concentrations of Zn2+ in artificial cerebrospinal fluid.

PubMed

Tamano, Haruna; Nishio, Ryusuke; Shakushi, Yukina; Sasaki, Miku; Koike, Yuta; Osawa, Misa; Takeda, Atsushi

2017-02-17

Artificial cerebrospinal fluid (ACSF), i.e., brain extracellular medium, which includes Ca 2+ and Mg 2+ , but not other divalent cations such as Zn 2+ , has been used for in vitro and in vivo experiments. The present study deals with the physiological significance of extracellular Zn 2+ in ACSF. Spontaneous presynaptic activity is suppressed in the stratum lucidum of brain slices from young rats bathed in ACSF containing 10 nM ZnCl 2 , indicating that extracellular Zn 2+ modifies hippocampal presynaptic activity. To examine the in vivo action of 10 nM ZnCl 2 on long-term potentiation (LTP), the recording region was perfused using a recording electrode attached to a microdialysis probe. The magnitude of LTP was not modified in young rats by perfusion with ACSF containing 10 nM ZnCl 2 , compared to perfusion with ACSF without Zn 2+ , but attenuated by perfusion with ACSF containing 100 nM ZnCl 2 . Interestingly, the magnitude of LTP was not modified in aged rats even by perfusion with ACSF containing 100 nM ZnCl 2 , but enhanced by perfusion with ACSF containing 10 mM CaEDTA, an extracellular Zn 2+ chelator. The present study indicates that the basal levels of extracellular Zn 2+ , which are in the range of low nanomolar concentrations, are critical for synaptic activity and perhaps increased age-dependently.

In vitro and in vivo physiology of low nanomolar concentrations of Zn2+ in artificial cerebrospinal fluid

PubMed Central

Tamano, Haruna; Nishio, Ryusuke; Shakushi, Yukina; Sasaki, Miku; koike, Yuta; Osawa, Misa; Takeda, Atsushi

2017-01-01

Artificial cerebrospinal fluid (ACSF), i.e., brain extracellular medium, which includes Ca2+ and Mg2+, but not other divalent cations such as Zn2+, has been used for in vitro and in vivo experiments. The present study deals with the physiological significance of extracellular Zn2+ in ACSF. Spontaneous presynaptic activity is suppressed in the stratum lucidum of brain slices from young rats bathed in ACSF containing 10 nM ZnCl2, indicating that extracellular Zn2+ modifies hippocampal presynaptic activity. To examine the in vivo action of 10 nM ZnCl2 on long-term potentiation (LTP), the recording region was perfused using a recording electrode attached to a microdialysis probe. The magnitude of LTP was not modified in young rats by perfusion with ACSF containing 10 nM ZnCl2, compared to perfusion with ACSF without Zn2+, but attenuated by perfusion with ACSF containing 100 nM ZnCl2. Interestingly, the magnitude of LTP was not modified in aged rats even by perfusion with ACSF containing 100 nM ZnCl2, but enhanced by perfusion with ACSF containing 10 mM CaEDTA, an extracellular Zn2+ chelator. The present study indicates that the basal levels of extracellular Zn2+, which are in the range of low nanomolar concentrations, are critical for synaptic activity and perhaps increased age-dependently. PMID:28211543

HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

PubMed

Mehta, Kosha J; Farnaud, Sebastien; Patel, Vinood B

2017-10-01

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p < 0.04) and 8 g/L (p = 0.05) treatments. HAMP expression showed alternating elevations and increased upon 1 g/L (p < 0.05) and 5 g/L (p < 0.05). However, in the recombinant cells that showed higher intracellular iron levels than wild-type cells, HFE and HAMP expressions were elevated only at low 1 g/L treatment (p < 0.03) and were repressed at 2 g/L treatment (p < 0.03). Under holotransferrin-untreated conditions, the iron-loaded recombinant cells showed higher expressions of HFE (p < 0.03) and HAMP (p = 0.05) than wild-type cells. HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

The Role of Ventral Tegmental Area Gamma-Aminobutyric Acid in Chronic Neuropathic Pain after Spinal Cord Injury in Rats.

PubMed

Ko, Moon Yi; Jang, Eun Young; Lee, June Yeon; Kim, Soo Phil; Whang, Sung Hun; Lee, Bong Hyo; Kim, Hee Young; Yang, Chae Ha; Cho, Hee Jung; Gwak, Young S

2018-04-20

Spinal cord injury (SCI) frequently results in chronic neuropathic pain (CNP). However, the understanding of brain neural circuits in CNP modulation is unclear. The present study examined the changes of ventral tegmental area (VTA) putative GABAergic and dopaminergic neuronal activity with CNP attenuation in rats. SCI was established by T10 clip compression injury (35 g, 1 min) in rats, and neuropathic pain behaviors, in vivo extracellular single-cell recording of putative VTA gamma-aminobutyric acid (GABA)/dopamine neurons, extracellular GABA level, glutamic acid decarboxylase (GAD), and vesicular GABA transporters (VGATs) were measured in the VTA, respectively. The results revealed that extracellular GABA level was significantly increased in the CNP group (50.5 ± 18.9 nM) compared to the sham control group (10.2 ± 1.7 nM). In addition, expression of GAD 65/67 , c-Fos, and VGAT exhibited significant increases in the SCI groups compared to the sham control group. With regard to neuropathic pain behaviors, spontaneous pain measured by ultrasound vocalizations (USVs) and evoked pain measured by paw withdrawal thresholds showed significant alteration, which was reversed by intravenous (i.v.) administration of morphine (0.5-5.0 mg/kg). With regard to in vivo electrophysiology, VTA putative GABAergic neuronal activity (13.6 ± 1.7 spikes/sec) and putative dopaminergic neuronal activity (2.4 ± 0.8 spikes/sec) were increased and decreased, respectively, in the SCI group compared to the sham control group. These neuronal activities were reversed by i.v. administration of morphine. The present study suggests that chronic increase of GABAergic neuronal activity suppresses dopaminergic neuronal activity in the VTA and is responsible for negative emotion and motivation for attenuation of SCI-induced CNP.

Baseline extracellular potassium level as an indicator of the rate of increase of the same on further storage in CPDA-1 whole blood units: a potential approach to complement FIFO system for prioritisation of blood bags for release from blood-banks.

PubMed

Baliarsingh, S; Jaiswal, M

2014-02-01

Potassium levels in stored blood bags increases as they age. Hyperkalemia in transfused blood has undesirable cardiac effects. Within a 19-month period, baseline and weekly samples from 15 CPDA-1 whole blood bags were collected till 28 days of storage and analysed for potassium, sodium, uric acid, albumin and whole blood haemoglobin. One unit increase in baseline (0 day) potassium in extracellular fluid of blood units was associated with the following increases in potassium levels on later days of storage: around two unit increase at 1 week (r2 = 0·50, P < 0·01) of storage; four units increase at 2 weeks (r2 = 0·64, P < 0·001) and 3 weeks (r2 = 0·51, P < 0·01) of storage; six units at 4 weeks (r2 = 0·53, P < 0·01) of storage. Baseline whole blood haemoglobin showed a moderate association with baseline potassium (r2 = 0·36, P < 0·05) and 2-week potassium (r2 = 0·35, P < 0·05) values. For CPDA-1 blood bags (i) low baseline potassium blood bags might be preferred for transfusion in cases demanding a low potassium load and (ii) coordinating the ‘first-in-first-out’ (FIFO) policy with ‘early release of blood-bags with high initial potassium’ might be helpful in improving the release of suitable blood units from blood-banks.

Cellular responses and bioremoval of nonylphenol by the bloom-forming cyanobacterium Planktothrix agardhii 1113

NASA Astrophysics Data System (ADS)

Medvedeva, Nadezda; Zaytseva, Tatyana; Kuzikova, Irina

2017-07-01

Nonylphenol (NP) is extensively used in agricultural, industrial and household applications. Moreover, NP is the major breakdown product of the nonionic surfactants, nonylphenol ethoxylates (NPEOs), the most widely used group of surfactants. Nonylphenol is persistent in the environment, highly toxic to aquatic organisms and is a potential endocrine disruptor. NP and NPEOs have been identified as priority hazardous substances under the Environmental Quality Standards Directive 2013/39/EU and are referred to in the list of substances of particular risk to the Baltic Sea. The toxicity of NP to the bloom-forming cyanobacterium Planktothrix agardhii 1113 isolated from the eastern Gulf of Finland, Baltic Sea and the bioremoval of NP by P. agardhii were studied. NP in concentrations > 0.4 mg L- 1 suppressed cyanobacterial growth. The median effective concentration of NP for P. agardhii after 4 days of treatment (EC50) was 1.5 mg L- 1. The removal of NP from the culture medium was primarily due to abiotic processes and biodegradation by the cyanobacterium rather than sorption by the cells. NP significantly increased the photosynthetic pigments, extracellular proteins and soluble exopolysaccharides content. The cyanobacterial growth inhibition was accompanied by the increased synthesis of microcystin dm-RR and of the odorous metabolites, geosmin and 2-methylisoborneol (MIB), by P. agardhii 1113. NP also notably increased the microcystin released into the environment. Increased levels of extracellular proteins, soluble exopolysaccharides, microcystins and odorous metabolites may affect the microbial loop in aquatic ecosystems. An increased level of malondialdehyde (MDA) was indicative of the formation of free radicals in P. agardhii under NP stress, whereas increased levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and proline indicated the occurrence of a scavenging mechanism.

TRPV1: Contribution to Retinal Ganglion Cell Apoptosis and Increased Intracellular Ca2+ with Exposure to Hydrostatic Pressure

PubMed Central

Sappington, Rebecca M.; Sidorova, Tatiana; Long, Daniel J.; Calkins, David J.

2013-01-01

Purpose Elevated hydrostatic pressure induces retinal ganglion cell (RGC) apoptosis in culture. The authors investigated whether the transient receptor potential vanilloid 1 (TRPV1) channel, which contributes to pressure sensing and Ca2+-dependent cell death in other systems, also contributes to pressure-induced RGC death and whether this contribution involves Ca2+. Methods trpv1 mRNA expression in RGCs was probed with the use of PCR and TRPV1 protein localization through immunocytochemistry. Subunit-specific antagonism (iodo-resiniferatoxin) and agonism (capsaicin) were used to probe how TRPV1 activation affects the survival of isolated RGCs at ambient and elevated hydrostatic pressure (+70 mm Hg). Finally, for RGCs under pressure, the authors tested whether EGTA chelation of Ca2+ improves survival and whether, with the Ca2+ dye Fluo-4 AM, TRPV1 contributes to increased intracellular Ca2+. Results RGCs express trpv1 mRNA, with robust TRPV1 protein localization to the cell body and axon. For isolated RGCs under pressure, TRPV1 antagonism increased cell density and reduced apoptosis to ambient levels (P ≤ 0.05), whereas for RGCs at ambient pressure, TRPV1 agonism reduced density and increased apoptosis to levels for elevated pressure (P ≤ 0.01). Chelation of extracellular Ca2+ reduced RGC apoptosis at elevated pressure by nearly twofold (P ≤ 0.01). Exposure to elevated hydrostatic pressure induced a fourfold increase in RGC intracellular Ca2+ that was reduced by half with TRPV1 antagonism. Finally, in the DBA/2 mouse model of glaucoma, levels of TRPV1 in RGCs increased with elevated IOP. Conclusions RGC apoptosis induced by elevated hydrostatic pressure arises substantially through TRPV1, likely through the influx of extracellular Ca2+. PMID:18952924

Modulation of sibutramine-induced increases in extracellular noradrenaline concentration in rat frontal cortex and hypothalamus by α2-adrenoceptors

PubMed Central

Wortley, K E; Heal, D J; Stanford, S C

1999-01-01

The effects of sibutramine (0.25–10 mg kg−1 i.p.) on extracellular noradrenaline concentration in the frontal cortex and hypothalamus of freely-moving rats were investigated using microdialysis. The role of presynaptic α2-adrenoceptors in modulating the effects of sibutramine in these brain areas was also determined.Sibutramine induced an increase in extracellular noradrenaline concentration, the magnitude of which paralleled dose, in both brain areas. In the cortex, this increase was gradual and sustained, whereas in the hypothalamus it was more rapid and of shorter duration.In both the cortex and hypothalamus, pretreatment of rats with the α2-adrenoceptor antagonist RX821002 (3 mg kg−1 i.p.) potentiated increases in the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.), by 7 and 10 fold respectively. RX821002 also reduced the latency of sibutramine to reach its maximum effect in the cortex, but not in the hypothalamus.Infusion of RX821002 (1 μM) via the probe increased the accumulation of extracellular noradrenaline induced by sibutramine (10 mg kg−1 i.p.) in both brain areas. In the hypothalamus, the effects of RX821002 on the accumulation of noradrenaline induced by sibutramine were 2 fold greater than those in the cortex.These findings support evidence that sibutramine inhibits the reuptake of noradrenaline in vivo, but that the accumulation of extracellular noradrenaline is limited by noradrenergic activation of presynaptic α2-adrenoceptors. Furthermore, the data suggest that terminal α2-adrenoceptors in the hypothalamus exert a greater inhibitory effect over the control of extracellular noradrenaline accumulation than do those in the cortex. PMID:10516646

Cardiac microvascular endothelial cells express a functional Ca+ -sensing receptor.

PubMed

Berra Romani, Roberto; Raqeeb, Abdul; Laforenza, Umberto; Scaffino, Manuela Federica; Moccia, Francesco; Avelino-Cruz, Josè Everardo; Oldani, Amanda; Coltrini, Daniela; Milesi, Veronica; Taglietti, Vanni; Tanzi, Franco

2009-01-01

The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores. Copyright 2008 S. Karger AG, Basel.

Protein disulfide isomerase externalization in endothelial cells follows classical and unconventional routes.

PubMed

Araujo, Thaís L S; Zeidler, Julianna D; Oliveira, Percíllia V S; Dias, Matheus H; Armelin, Hugo A; Laurindo, Francisco R M

2017-02-01

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions. Copyright © 2016 Elsevier Inc. All rights reserved.

P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain.

PubMed

Potschka, H; Fedrowitz, M; Löscher, W

2001-11-16

Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice

PubMed Central

Dadson, Keith; Turdi, Subat; Boo, Stellar; Hinz, Boris; Sweeney, Gary

2015-01-01

Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM) remodeling in response to pressure overload (PO). Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO) and wild-type (WT) mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson’s trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects. PMID:25910275

A Phex Mutation in a Murine Model of X-linked Hypophosphatemia Alters Phosphate Responsiveness of Bone Cells

PubMed Central

Ichikawa, Shoji; Austin, Anthony M.; Gray, Amie K.; Econs, Michael J.

2011-01-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH – high dose phosphate and calcitriol – further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (PhexK496X) and hyperphosphatemic tumoral calcinosis (Galnt3 -/-), and Galnt3/Phex double mutant mice. Phex mutant mice had not only increased Fgf23 expression, but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by up-regulating Fgf23 expression as much as 24 fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for “normal” phosphate levels. PMID:22006791

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Econs, Michael J

2012-02-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.

PubMed

Corrêa, Gladys; Almeida Lindenberg, Carolina de; Moreira-Souza, Aline Cristina de Abreu; Savio, Luiz Eduardo Baggio; Takiya, Christina Maeda; Marques-da-Silva, Camila; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

2017-04-01

Activation of the purinergic P2X7 receptor by extracellular ATP (eATP) potentiates proinflammatory responses during infections by intracellular pathogens. Extracellular ATP triggers an antimicrobial response in macrophages infected with Toxoplasma gondii in vitro, suggesting that purinergic signaling may stimulate host defense mechanisms against toxoplasmosis. Here, we provide in vivo evidence in support of this hypothesis, by showing that P2X7 -/- mice are more susceptible than P2X7 +/+ mice to acute infection by the RH strain of T. gondii, and that this phenomenon is associated with a deficient proinflammatory response. Four days post-infection, peritoneal washes from infected P2X7 -/- mice had no or little increase in the levels of the proinflammatory cytokines IL-12, IL-1β, IFN-γ, and TNF-α, whose levels increased markedly in samples from infected P2X7 +/+ mice. Infected P2X7 -/- mice displayed an increase in organ weight and histological alterations in some of the 'shock organs' in toxoplasmosis - the liver, spleen and mesenteric lymph nodes. The liver of infected P2X7 -/- mice had smaller granulomas, but increased parasite load/granuloma. Our results confirm that the P2X7 receptor is involved in containing T. gondii spread in vivo, by stimulating inflammation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

PubMed Central

Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

2016-01-01

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

Promotion of Iron Oxide Reduction and Extracellular Electron Transfer in Shewanella oneidensis by DMSO

PubMed Central

Cheng, Yuan-Yuan; Li, Bing-Bing; Li, Dao-Bo; Chen, Jie-Jie; Li, Wen-Wei; Tong, Zhong-Hua; Wu, Chao; Yu, Han-Qing

2013-01-01

The dissimilatory metal reducing bacterium Shewanella oneidensis MR-1, known for its capacity of reducing iron and manganese oxides, has great environmental impacts. The iron oxides reducing process is affected by the coexistence of alternative electron acceptors in the environment, while investigation into it is limited so far. In this work, the impact of dimethyl sulphoxide (DMSO), a ubiquitous chemical in marine environment, on the reduction of hydrous ferric oxide (HFO) by S. oneidensis MR-1 was investigated. Results show that DMSO promoted HFO reduction by both wild type and ΔdmsE, but had no effect on the HFO reduction by ΔdmsB, indicating that such a promotion was dependent on the DMSO respiration. With the DMSO dosing, the levels of extracellular flavins and omcA expression were significantly increased in WT and further increased in ΔdmsE. Bioelectrochemical analysis show that DMSO also promoted the extracellular electron transfer of WT and ΔdmsE. These results demonstrate that DMSO could stimulate the HFO reduction through metabolic and genetic regulation in S. oneidensis MR-1, rather than compete for electrons with HFO. This may provide a potential respiratory pathway to enhance the microbial electron flows for environmental and engineering applications. PMID:24244312

Dysregulation of Corticostriatal Ascorbate Release and Glutamate Uptake in Transgenic Models of Huntington's Disease

PubMed Central

2013-01-01

Abstract Significance: Dysregulation of cortical and striatal neuronal processing plays a critical role in Huntington's disease (HD), a dominantly inherited condition that includes a progressive deterioration of cognitive and motor control. Growing evidence indicates that ascorbate (AA), an antioxidant vitamin, is released into striatal extracellular fluid when glutamate is cleared after its release from cortical afferents. Both AA release and glutamate uptake are impaired in the striatum of transgenic mouse models of HD owing to a downregulation of glutamate transporter 1 (GLT1), the protein primarily found on astrocytes and responsible for removing most extracellular glutamate. Improved understanding of an AA–glutamate interaction could lead to new therapeutic strategies for HD. Recent Advances: Increased expression of GLT1 following treatment with ceftriaxone, a beta-lactam antibiotic, increases striatal glutamate uptake and AA release and also improves the HD behavioral phenotype. In fact, treatment with AA alone restores striatal extracellular AA to wild-type levels in HD mice and not only improves behavior but also improves the firing pattern of neurons in HD striatum. Critical Issues: Although evidence is growing for an AA-glutamate interaction, several key issues require clarification: the site of action of AA on striatal neurons; the precise role of GLT1 in striatal AA release; and the mechanism by which HD interferes with this role. Future Directions: Further assessment of how the HD mutation alters corticostriatal signaling is an important next step. A critical focus is the role of astrocytes, which express GLT1 and may be the primary source of extracellular AA. Antioxid. Redox Signal. 19, 2115–2128. PMID:23642110

Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.

PubMed

Yoo, Dae-goon; Winn, Matthew; Pang, Lan; Moskowitz, Samuel M; Malech, Harry L; Leto, Thomas L; Rada, Balázs

2014-05-15

Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust neutrophil recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection. Neutrophil primary granule components, myeloperoxidase (MPO) and human neutrophil elastase (HNE), are inflammatory markers in CF airways, and their increased levels are associated with poor lung function. Identifying the mechanism of MPO and HNE release from neutrophils is of high clinical relevance for CF. In this article, we show that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. Bacteria are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory standard strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. The increase in peroxidase activity of neutrophil supernatants after Pseudomonas exposure indicates that enzymatically active MPO is released. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release. Inhibition of the cytoskeleton prevents Pseudomonas-initiated superoxide production and DNA release. NADPH oxidase inhibition suppresses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas. Our data describe in vitro details of DNA, MPO, and HNE release from neutrophils activated by P. aeruginosa. We propose that Pseudomonas-induced NET formation is an important mechanism contributing to inflammatory conditions characteristic of CF airways.

The effects of nitric oxide-cGMP pathway stimulation on dopamine in the medial preoptic area and copulation in DHT-treated castrated male rats

PubMed Central

Sato, Satoru M.; Wersinger, Scott R.; Hull, Elaine M.

2007-01-01

Dopamine (DA) in the medial preoptic area (MPOA) provides important facilitative influence on male rat copulation. We have shown that the nitric oxide-cGMP (NO-cGMP) pathway modulates MPOA DA levels and copulation. We have also shown that systemic estradiol (E2) maintains neuronal NO synthase (nNOS) immunoreactivity in the MPOA of castrates, as well as relatively normal DA levels. This effect of E2 on nNOS probably accounts for at least some of the previously demonstrated behavioral facilitation by intra-MPOA E2 administration in castrates. Therefore, we hypothesized that stimulation of the MPOA NO-cGMP pathway in dihydrotestosterone (DHT)-treated castrates should restore DA levels and copulatory behaviors. Reverse-dialysis of a NO donor, sodium nitroprusside (SNP), increased extracellular DA in the MPOA of DHT-treated castrates and restored the ability to copulate to ejaculation in half of the animals. A cGMP analog, 8-Br-cGMP, also increased extracellular DA, though not as robustly, but did not restore copulatory ability. The effectiveness of the NO donor in restoring copulation and MPOA DA levels is consistent with our hypothesis. However, the lack of behavioral effects of 8-Br-cGMP, despite its increase in MPOA DA, suggests that NO may have additional mediators in the MPOA in the regulation of copulation. Furthermore, the suboptimal copulation seen in the NO donor-treated animals suggests the importance of extra-MPOA systems in the regulation of copulation. PMID:17467707

Local radiotherapy increases the level of autoantibodies to ribosomal P0 protein but not to heat shock proteins, extracellular matrix molecules and EGFR/ErbB2 receptors in prostate cancer patients.

PubMed

Ingrosso, Gianluca; Fantini, Massimo; Nardi, Alessandra; Benvenuto, Monica; Sacchetti, Pamela; Masuelli, Laura; Ponti, Elisabetta; Frajese, Giovanni Vanni; Lista, Florigio; Schillaci, Orazio; Santoni, Riccardo; Modesti, Andrea; Bei, Roberto

2013-03-01

Prostate cancer is a common cancer among men in developed countries. Although hormonotherapy and radiotherapy (RT) represent valid therapies for prostate cancer treatment, novel immunological approaches have been explored. The development of clinical trials employing cancer vaccines has indicated that immune response to tumor antigens can be boosted and that vaccine administration can improve patient survival. Immune response to tumor antigens could also be enhanced after standard therapies. In the present study, we determined the occurrence of antibodies to extracellular matrix (ECM) molecules, heat shock protein (HSP), ribosomal P0 protein, EGFR, ErbB2 and prostate-specific antigen (PSA) in 35 prostate cancer patients prior to and following local RT and hormonotherapy. We demonstrated that immunity to P0, ECM molecules [collagens (C) CI, CIII, CV, fibronectin (FN) and laminin (LM)] and to HSP90 was associated with malignancy in untreated patients. None of the patient sera showed antibodies to EGFR, while 2 and 1 patients showed reactivity to ErbB2 and PSA, respectively. We also demonstrated that 8 months after therapy the IgG serum levels to CI, CIII, FN and HSP90 significantly decreased. Conversely, the level of P0 autoantibodies increased after therapy in 10 patients. Five of the 10 patients with increased levels of P0 autoantibodies were treated with RT plus hormonotherapy. Treatment of patients did not change the levels of antibodies against EGFR, ErbB2 and PSA. Our results indicated that the modification of antibody level to self molecules after standard treatment of prostate cancer patients is influenced by the type of antigen. Ribosomal P0 protein appears to be a high immunogenic antigen and its immunogenicity increases following RT. In addition, 10 patients with increased levels of autoantibodies to P0 showed PSA mean levels lower than the remaining 25 patients at 18 months. This study may contribute to a better understanding of the immunobiological behavior of prostate cancer patients following standard treatment.

Quercetin attenuates mitochondrial dysfunction and biogenesis via upregulated AMPK/SIRT1 signaling pathway in OA rats.

PubMed

Qiu, Linan; Luo, Yuju; Chen, Xiaojuan

2018-07-01

Despite the severity of osteoarthritis (OA), current medical therapy strategies for OA aim at symptom control and pain reduction, as there is no ideal drug for effective OA treatment. OA rat model was used to explore the therapeutic function of quercetin on remission of OA, by determining the reactive oxygen species (ROS) levels, mitochondrial function and extracellular matrix integrity. Quercetin could attenuate ROS generation and augment the glutathione (GSH) and glutathione peroxidase (GPx) expression levels in OA rat. Quercetin not only enhanced mitochondrial membrane potential, oxygen consumption, adenosine triphosphate (ATP) levels in mitochondria, but also increased the mitochondrial copy number. Furthermore, the interlukin (IL)-1β-induced accumulation of nitric oxide (NO), matrixmetalloproteinase (MMP)-3) and MMP-13 could be suppressed by quercetin. Finally, we confirmed that the therapeutic properties of quercetin on OA might function through the adenosine monophosphate-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway. In summary, quercetin could alleviate OA through attenuating the ROS levels, reversing the mitochondrial dysfunction and keeping the integrality of extracellular matrix of joint cartilage. The underlying mechanism might involve the regulation of AMPK/SIRT1 signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The vigilance promoting drug modafinil increases extracellular glutamate levels in the medial preoptic area and the posterior hypothalamus of the conscious rat: prevention by local GABAA receptor blockade.

PubMed

Ferraro, L; Antonelli, T; Tanganelli, S; O'Connor, W T; Perez de la Mora, M; Mendez-Franco, J; Rambert, F A; Fuxe, K

1999-04-01

The effects of modafinil on glutamatergic and GABAergic transmission in the rat medial preoptic area (MPA) and posterior hypothalamus (PH), are analysed. Modafinil (30-300 mg/kg) increased glutamate and decreased GABA levels in the MPA and PH. Local perfusion with the GABAA agonist muscimol (10 microM), reduced, while the GABAA antagonist bicuculline (1 microM and 10 microM) increased glutamate levels. The modafinil (100 mg/kg)-induced increase of glutamate levels was antagonized by local perfusion with bicuculline (1 microM). When glutamate levels were increased by the local perfusion with the glutamate uptake inhibitor L-trans-PDC (0.5 mM), modafinil produced an additional enhancement of glutamate levels. Modafinil (1-33 microM) failed to affect [3H]glutamate uptake in hypothalamic synaptosomes and slices. These findings show that modafinil increases glutamate and decreases GABA levels in MPA and PH. The evidence that bicuculline counteracts the modafinil-induced increase of glutamate levels strengthens the evidence for an inhibitory GABA/glutamate interaction in the above regions controlling the sleep-wakefulness cycle.

Heparin Stimulates Elastogenesis: Application to Silk-Based Vascular Grafts

PubMed Central

Baughman, Cassandra; Kaplan, David L.; Castellot, John J.

2013-01-01

With over 500,000 coronary artery bypass grafts (CABG) performed annually in the United States alone, there is a significant clinical need for a small diameter tissue engineered vascular graft. A principle goal in tissue engineering is to develop materials and growth conditions that encourage appropriate re-cellularization and extracellular matrix formation in vivo. A particular challenge in vascular tissue engineering results from the inability of adult cells to produce elastin, as its expression is developmentally limited. We investigated factors to stimulate elastogenesis in vitro, and found that heparin treatment of adult human vascular smooth muscle cells promoted the formation of elastic fibers. This effect was heparin-specific, and dependent on cell density and growth state. We then applied this information to a silk-based construct, and found that immobilized heparin showed essentially identical biological effects to that of soluble heparin. These findings indicate that heparinized vascular grafts may promote elastin formation and regulate restenosis, in addition to heparin’s well-established antithrombotic properties. Given the increase in elastin mRNA level and the increase in extracellular elastin present, our data suggests that there may be multiple levels of elastin regulation that are mediated by heparin treatment. PMID:21600981

Forced swimming test and fluoxetine treatment: in vivo evidence that peripheral 5-HT in rat platelet-rich plasma mirrors cerebral extracellular 5-HT levels, whilst 5-HT in isolated platelets mirrors neuronal 5-HT changes.

PubMed

Bianchi, M; Moser, C; Lazzarini, C; Vecchiato, E; Crespi, F

2002-03-01

Low levels of central serotonin (5-HT) have been related to the state of depression, and 5-HT is the major target of the newer antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs). Neurons and platelets display structural and functional similarities, so that the latter have been proposed as a peripheral model of central functions. In particular, in blood more than 99% of 5-HT is contained in platelets, so that one could consider changes in 5-HT levels in platelets as a mirror of changes in central 5-HT. Here, this hypothesis has been studied via the analysis of the influence of: (1) the forced swimming test (FST, which has been proved to be of utility to predict the clinical efficacy of antidepressants in rodents) and (2) treatment with the SSRI fluoxetine upon 5-HT levels monitored in brain regions and in peripheral platelets by means of electrochemical in vivo and ex vivo measurements. The results obtained confirm that the FST increases immobility; furthermore they show a parallel and significant decrease in cerebral (brain homogenate) and peripheral (in platelet-rich plasma, PRP) voltammetric 5-HT levels following the FST in naive rats. In addition, subchronic treatment with fluoxetine was followed by a significant increase in 5-HT levels in PRP, while the same SSRI treatment performed within the FST resulted in a decrease in the 5-HT levels in PRP. However, this decrease was inferior to that observed without SSRI treatment. These data suggest that there is an inverse relationship between immobility and the levels of 5-HT in PRP and that these peripheral 5-HT levels are sensitive to: (1) the FST, (2) the treatment with fluoxetine and (3) the combination of both treatments, i.e. SSRI + FST. It has been reported that SSRI treatment at first inhibits the 5-HT transporter in brain, resulting in increased extracellular 5-HT, while following sustained SSRI treatments decreased intracellular levels of central 5-HT were observed. Accordingly, the present data show that the initial block of 5-HT reuptake is revealed by the selective increase in 5-HT levels (extracellular content) measured in PRP (not in insulated platelets, IPs) the 1st day of fluoxetine treatment. The initial action of this SSRI upon the 5-HT transporter in brain has also been confirmed by in vivo voltammetric data showing selective increase in the serotonergic signal following local injection of fluoxetine into the brain region studied. Successively, the major effect monitored is a decrease in 5-HT levels, which is more evident in IPs than in PRP. However, it is known that following 2 weeks treatment with an SSRI, 5-HT autoreceptors are desensitized and the serotonin synthesis is restored, together with the intracellular 5-HT levels. The present data showing that the levels of 5-HT in IPs tend to return to control values 12 days after the beginning of chronic fluoxetine treatment suggest that 5-HT levels in IPs (intracellular environment) mirror the influence of SSRI treatment upon the central 5-HT system. On the other hand, at day 12 of the chronic fluoxetine treatment, 5-HT content remains low in PRP. Similarly, low levels of 5-HT have been monitored in brain homogenate of rats chronically treated with fluoxetine. This would support the similarity between PRP preparation and brain homogenate as in both cases cells are disrupted by sample preparation. In conclusion this work supports the literature in proposing platelets as a peripheral model of central functions. In particular, the present data support the idea that peripheral 5-HT platelet levels can reflect the state of the central 5-HT system in conditions of depression. Furthermore, the main outcome of this study is that PRP may mirror central extracellular 5-HT levels, whilst IPs mirror neuronal 5-HT changes.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel.

PubMed

Stroffekova, K; Kupert, E Y; Malinowska, D H; Cuppoletti, J

1998-10-01

Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.

[Effect of electroacupuncture intervention on expression of extracellular matrix collagen and metabolic enzymes].

PubMed

Liao, Jun; Zhang, Le; Ke, Mei-gui; Xu, Teng

2013-12-01

To observe the effect of electroacupuncture (EA) at "Dazhui" (GV 14) on the contents of extracellular matrix (ECM), collagen type II (COL-II), collagen type V (COL-V), matrix metalloproteinase (MMP)-13, tissue inhibitor of metalloproteinase (TIMP)-1 in rats with cervicovertebral disc degeneration so as to explore its mechanism underlying relief of intervertebral disc degeneration. A total of 28 SD rats were randomly divided into sham group (n = 7), model group (n = 7), EA group (n = 7) and medication group (n = 7). The model of cervical intervertebral disc degeneration was established by trans-section of the deep neck splenius, the longest muscles of head, neck costocervicalis, head semi-spinatus muscle, supraspinous ligament and interspinal ligaments of cervical 2-7 segments, etc. to produce imbalance between the dynamic and static force. EA was applied to "Dazhui" (GV 14) for 30 min, once daily for 28 days, with a 2 days' interval between two courses. Animals of the medication group were treated by oral administration of meloxicam tablets (0.75 mg/kg) once daily for 28 days, with a 2 days' interval between two courses. Immunohistochemistry was used to measure the expression of ECM, COL- II, COL-V, MMP-13 and TIMP-1 in the cervicovertebral disc tissue. Compared with the sham group, the expression levels of ECM and COL-II proteins in the cervicovertebral disc tissue were significantly decreased in the model group (P < 0.01), while COL-V and MMP-13 expression levels in the model group were significantly increased (P < 0.01, P < 0.05). Compared with the model group, both ECM and COL-Il expression levels were considerably increased in the EA group and medication group (P < 0.01), while COL-V and MMP-13 expression levels were considerably down-regulated (P < 0.01, P < 0.05). No significant differences were found among the four groups in TIMP-1 expression levels (P > 0.05). EA of "Dazhui" (GV 14) can effectively regulate extracellular matrix system in rats with cervical intervertebral disc degeneration, which is possibly related to its effect in relieving cervical spondylosis.

Regulation of aortic extracellular matrix synthesis via noradrenergic system and angiotensin II in juvenile rats.

PubMed

Dab, Houcine; Hachani, Rafik; Dhaouadi, Nedra; Sakly, Mohsen; Hodroj, Wassim; Randon, Jacques; Bricca, Giampiero; Kacem, Kamel

2012-10-01

Extracellular matrix (ECM) synthesis regulation by sympathetic nervous system (SNS) or angiotensin II (ANG II) was widely reported, but interaction between the two systems on ECM synthesis needs further investigation. We tested implication of SNS and ANG II on ECM synthesis in juvenile rat aorta. Sympathectomy with guanethidine (50 mg/kg, subcutaneous) and blockade of the ANG II AT1 receptors (AT1R) blocker with losartan (20 mg/kg/day in drinking water) were performed alone or in combination in rats. mRNA and protein synthesis of collagen and elastin were examined by Q-RT-PCR and immunoblotting. Collagen type I and III mRNA were increased respectively by 62 and 43% after sympathectomy and decreased respectively by 31 and 60% after AT1R blockade. Combined treatment increased collagen type III by 36% but not collagen type I. The same tendency of collagen expression was observed at mRNA and protein levels after the three treatments. mRNA and protein level of elastin was decreased respectively by 63 and 39% and increased by 158 and 15% after losartan treatment. Combined treatment abrogates changes induced by single treatments. The two systems act as antagonists on ECM expression in the aorta and combined inhibition of the two systems prevents imbalance of mRNA and protein level of collagen I and elastin induced by single treatment. Combined inhibition of the two systems prevents deposit or excessive reduction of ECM and can more prevent cardiovascular disorders.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

PubMed

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis. PMID:26273425

Caffeine Induces a Stimulant Effect and Increases Dopamine Release in the Nucleus Accumbens Shell Through the Pulmonary Inhalation Route of Administration in Rats.

PubMed

Galvalisi, Martín; Prieto, José Pedro; Martínez, Marcela; Abin-Carriquiry, Juan Andrés; Scorza, Cecilia

2017-01-01

Oral, intraperitoneal, or intravenous have been the common routes of administration used to study the behavioral and neurochemical pharmacology of caffeine, one of the most widely used psychoactive substances worldwide. We have reported that caffeine is an active adulterant frequently found in coca-paste (CP)-seized samples, a highly addictive form of smokable cocaine. The role of caffeine in the psychostimulant and neurochemical effects induced by CP remains under study. No preclinical animal studies have been performed so far to characterize the effects of caffeine when it is administered through the pulmonary inhalation route. Caffeine (10, 25, and 50 mg) was volatilized and rats were exposed to one inhalation session of its vapor. The stimulant effect was automatically recorded and plasmatic levels of caffeine were measured. Caffeine capability (50 mg) to increase extracellular dopamine (DA) levels in nucleus accumbens shell was also studied by in vivo microdialysis in non-anesthetized animals. A dose-dependent stimulant effect induced by volatilized caffeine was observed and this effect was directly related with caffeine plasmatic levels. A significant increase in the extracellular DA was achieved after 50 mg of volatilized caffeine exposure. This is the first report showing pharmacological acute effects of caffeine through the pulmonary inhalation route of administration and suggests that this could be a condition under which caffeine can elevate its weak reinforcing effect and even enhance the psychostimulant effect and abuse liability of smokable adulterated psychostimulant drugs.

The Extracellular Matrix Protein Brevican Limits Time-Dependent Enhancement of Cocaine Conditioned Place Preference.

PubMed

Lubbers, Bart R; Matos, Mariana R; Horn, Annemarie; Visser, Esther; Van der Loo, Rolinka C; Gouwenberg, Yvonne; Meerhoff, Gideon F; Frischknecht, Renato; Seidenbecher, Constanze I; Smit, August B; Spijker, Sabine; van den Oever, Michel C

2016-06-01

Cocaine-associated environmental cues sustain relapse vulnerability by reactivating long-lasting memories of cocaine reward. During periods of abstinence, responding to cocaine cues can time-dependently intensify a phenomenon referred to as 'incubation of cocaine craving'. Here, we investigated the role of the extracellular matrix protein brevican in recent (1 day after training) and remote (3 weeks after training) expression of cocaine conditioned place preference (CPP). Wild-type and Brevican heterozygous knock-out mice, which express brevican at ~50% of wild-type levels, received three cocaine-context pairings using a relatively low dose of cocaine (5 mg/kg). In a drug-free CPP test, heterozygous mice showed enhanced preference for the cocaine-associated context at the remote time point compared with the recent time point. This progressive increase was not observed in wild-type mice and it did not generalize to contextual-fear memory. Virally mediated overexpression of brevican levels in the hippocampus, but not medial prefrontal cortex, of heterozygous mice prevented the progressive increase in cocaine CPP, but only when overexpression was induced before conditioning. Post-conditioning overexpression of brevican did not affect remote cocaine CPP, suggesting that brevican limited the increase in remote CPP by altering neuro-adaptive mechanisms during cocaine conditioning. We provide causal evidence that hippocampal brevican levels control time-dependent enhancement of cocaine CPP during abstinence, pointing to a novel substrate that regulates incubation of responding to cocaine-associated cues.

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

PubMed Central

Takeda, Atsushi; Nakamura, Masatoshi; Fujii, Hiroaki; Uematsu, Chihiro; Minamino, Tatsuya; Adlard, Paul A.; Bush, Ashley I.; Tamano, Haruna

2014-01-01

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn2+ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently Aβ-induced cognition deficits. PMID:25536033

Amyloid β-mediated Zn2+ influx into dentate granule cells transiently induces a short-term cognitive deficit.

PubMed

Takeda, Atsushi; Nakamura, Masatoshi; Fujii, Hiroaki; Uematsu, Chihiro; Minamino, Tatsuya; Adlard, Paul A; Bush, Ashley I; Tamano, Haruna

2014-01-01

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn2+ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn2+ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn2+ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn2+ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn2+ levels in the dentate granule cell layer in ACSF without Zn2+, but in ACSF containing Zn2+. The increase in intracellular Zn2+ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn2+ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn2+ may play a key role for transiently Aβ-induced cognition deficits.

Increases in extracellular zinc in the amygdala in acquisition and recall of fear experience and their roles in response to fear.

PubMed

Takeda, A; Tamano, H; Imano, S; Oku, N

2010-07-14

The amygdala is enriched with histochemically reactive zinc, which is dynamically coupled with neuronal activity and co-released with glutamate. The dynamics of the zinc in the amygdala was analyzed in rats, which were subjected to inescapable stress, to understand the role of the zinc in emotional behavior. In the communication box, two rats were subjected to foot shock stress and anxiety stress experiencing emotional responses of foot-shocked rat under amygdalar perfusion. Extracellular zinc was increased by foot shock stress, while decreased by anxiety stress, suggesting that the differential changes in extracellular zinc are associated with emotional behavior. In rats conditioned with foot shock, furthermore, extracellular zinc was increased again in the recall of fear (foot shock) in the same box without foot shock. When this recall was performed under perfusion with CaEDTA, a membrane-impermeable zinc chelator, to examine the role of the increase in extracellular zinc, the time of freezing behavior was more increased, suggesting that zinc released in the lateral amygdala during the recall of fear participates in freezing behavior. To examine the role of the increase in extracellular zinc during fear conditioning, fear conditioning was also performed under perfusion with CaEDTA. The time of freezing behavior was more increased in the contextual recall, suggesting that zinc released in the lateral nucleus during fear conditioning also participates in freezing behavior in the recall. In brain slice experiment, CaEDTA enhanced presynaptic activity (exocytosis) in the lateral nucleus after activation of the entorhinal cortex. The present paper demonstrates that zinc released in the lateral amygdala may participate in emotional behavior in response to fear. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

PubMed Central

Miller, B E; Hook, G E

1988-01-01

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids. PMID:2845927

Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

PubMed Central

Povero, Davide; Eguchi, Akiko; Li, Hongying; Johnson, Casey D.; Papouchado, Bettina G.; Wree, Alexander; Messer, Karen; Feldstein, Ariel E.

2014-01-01

Background & Aim Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. Design Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. Results We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. Conclusions These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD. PMID:25470250

Pharmacological activation of LXRs decreases amyloid-β levels in Niemann-Pick type C model cells.

PubMed

Stefulj, Jasminka; Peric, Maja; Malnar, Martina; Kosicek, Marko; Schweinzer, Cornelia; Zivkovic, Jelena; Scholler, Monika; Panzenboeck, Ute; Hecimovic, Silva

2013-01-01

Niemann-Pick type C disease (NPC) is an inherited disorder mainly caused by loss-of-function mutations in the NPC1 gene, that lead to intracellular cholesterol accumulation and disturbed cholesterol homeostasis. Similarly to Alzheimer's disease (AD), NPC is associated with progressive neurodegeneration and altered metabolism of amyloid precursor protein (APP). Liver X receptors (LXRs), the key transcriptional regulators of cholesterol homeostasis, were reported to play neuroprotective roles in NPC mice. We investigated the impacts of LXRs on APP metabolism in mutant CHO cells lacking the NPC1 gene (-NPC1 cells). Pharmacological activation of LXRs in -NPC1 cells tended to reduce the ratio of total secreted APP (sAPP) to full length APP (flAPP) levels and sAPPβ levels as well as to increase the ratio of APP Cterminal fragments to flAPP levels, resulting in decreased levels of amyloid β (Aβ) peptides. -NPC1 cells treated with LXR agonist TO901317 (TO90) displayed a modest increase in cholesterol efflux to apolipoprotein A-I (apoA-I) but not to HDL3, or in the absence of extracellular cholesterol acceptors. The observed similar reduction of Aβ levels upon TO90 treatment in the presence or in the absence of extracellular apoA-I indicated a cholesterol-efflux independent effect of TO90 on Aβ levels. Furthermore, TO90 had no effect on the cholesterol synthesis rate in -NPC1 cells, while it reduced the rate of cholesterol esterification. The obtained results indicate that LXR activation may decrease Aβ levels in NPC1- deficient conditions. The underlying mechanism of this action does not appear to be related to effects on cholesterol efflux or synthesis rates.

Striatal dopamine neurotransmission: regulation of release and uptake

PubMed Central

Sulzer, David; Cragg, Stephanie J.; Rice, Margaret E.

2016-01-01

Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients. PMID:27141430

A pharmacoproteomic study confirms the synergistic effect of chondroitin sulfate and glucosamine

PubMed Central

Calamia, Valentina; Mateos, Jesús; Fernández-Puente, Patricia; Lourido, Lucía; Rocha, Beatriz; Fernández-Costa, Carolina; Montell, Eulalia; Vergés, Josep; Ruiz-Romero, Cristina; Blanco, Francisco J.

2014-01-01

Osteoarthritis (OA) is the most common age-related rheumatic disease. Chondrocytes play a primary role in mediating cartilage destruction and extracellular matrix (ECM) breakdown, which are main features of the OA joint. Quantitative proteomics technologies are demonstrating a very interesting power for studying the molecular effects of some drugs currently used to treat OA patients, such as chondroitin sulfate (CS) and glucosamine (GlcN). In this work, we employed the iTRAQ (isobaric tags for relative and absolute quantitation) technique to assess the effect of CS and GlcN, both alone and in combination, in modifying cartilage ECM metabolism by the analysis of OA chondrocytes secretome. 186 different proteins secreted by the treated OA chondrocytes were identified. 36 of them presented statistically significant differences (p ≤ 0.05) between untreated and treated samples: 32 were increased and 4 decreased. The synergistic chondroprotective effect of CS and GlcN, firstly reported by our group at the intracellular level, is now demonstrated also at the extracellular level. PMID:24912619

Prostaglandin E1 inhibits collagen expression in anti-thymocyte antibody-induced glomerulonephritis: possible role of TGF beta.

PubMed

Schneider, A; Thaiss, F; Rau, H P; Wolf, G; Zahner, G; Jocks, T; Helmchen, U; Stahl, R A

1996-07-01

To test whether or not prostaglandins mediate extracellular matrix formation in immune-mediated glomerular disease, rats with anti-thymocyte antibody-induced glomerulonephritis were treated with prostaglandin E1 (PGE1) (250 micrograms/twice daily/s.c.). Glomerular expression of collagen types III and IV was assessed by Northern blotting, immunohistology and Western blotting. Proliferation of glomerular cells was evaluated by staining for the proliferating cell nuclear antigen (PCNA) and consecutive cell counting. At day five after induction of the disease, glomerular mRNA levels of collagen types III and IV were three- to fivefold higher compared with non-nephritic controls. Similarly glomerular deposition of these collagens was markedly increased when assessed by immunohistology. The treatment of nephritic rats with PGE1 reduced the increased glomerular mRNA levels as well as the protein concentration and the deposition of extracellular collagens. The number of PCNA positive cells which was significantly higher in nephritic rats when compared with control animals (24 hr, nephritis 2.53 +/- 0.33 and Control 0.26 +/- 0.06, P = 0.011; 5 days, nephritis 5.10 +/- 1.13 and Control 0.75 +/- 0.08, cells per glomerular cross section, P = 0.03) was reduced by PGE1 (24 hr, nephritis+PGE1 0.44 +/- 0.30, P = 0.0001; 5 days, nephritis +/- PGE1 1.91 +/- 1.84 cells per glomerular cross section, P = 0.001). Prostaglandin E1 also ameliorated the glomerular infiltration of monocytes at 24 hours (nephritis 4.36 +/- 2.82, nephritis + PGE1 2.20 +/- 1.82, cells per glomerular cross section) and five days (nephritis 1.51 +/- 0.58, nephritis+PGE1 1.12 +/- 0.61, cells per glomerular cross section). To further characterize possible mechanisms by which PGE1 reduces extracellular matrix deposition, the glomerular expression of transforming growth factor (TGF-beta), and interleukin 1 beta (IL-1 beta) was assessed by Northern blotting. Nephritic glomeruli showed increased mRNA levels of TGF-beta at day 5 and IL-1 beta at 24 hours when compared with control kidneys. Treatment of the animals with PGE1 inhibited the mRNA expression of TGF-beta and IL-1 beta. These data demonstrate that PGE1 reduces the glomerular expression of extracellular matrix proteins in anti-thymocyte antibody-induced glomerulonephritis, suggesting a beneficial role of prostaglandins in this proliferative glomerular immune injury. The effects of PGE1 might be mediated by inhibition of TGF-beta and IL-1 beta production.

Feeding-associated alterations in striatal neurotransmitter release

NASA Technical Reports Server (NTRS)

Acworth, I. N.; Ressler, K.; Wurtman, R. J.

1989-01-01

Published evidence suggests a role for dopaminergic (DA) brain pathways in feeding-associated behaviors. Using the novel technique of brain microdialysis of striatal extracellular fluid (ECF) as an index of DA release, Church et al. described increases in levels of DA when animals had limited access to pellets, but not with free access. Dopamine release from the nucleus accumbens did increase with free access to pellets post starvation or after food reward. We used permanently implanted microdialysis probes to measure ECF levels of DA, DOPAC, HVA, and large neutral amino acids (LNAA) for up to 72 hours after implantation among rats experiencing different dietary regimens.

Involvement of glucocorticoid-mediated Zn2+ signaling in attenuation of hippocampal CA1 LTP by acute stress.

PubMed

Takeda, Atsushi; Suzuki, Miki; Tamano, Haruna; Takada, Shunsuke; Ide, Kazuki; Oku, Naoto

2012-03-01

Glucocorticoid-glutamatergic interactions have been proposed as a potential model to explain stress-mediated impairment of cognition. However, it is unknown whether glucocorticoid-zincergic interactions are involved in this impairment. Histochemically reactive zinc (Zn(2+)) is co-released with glutamate from zincergic neurons. In the present study, involvement of synaptic Zn(2+) in stress-induced attenuation of CA1 LTP was examined in hippocampal slices from young rats after exposure to tail suspension stress for 30s, which significantly increased serum corticosterone. Stress-induced attenuation of CA1 LTP was ameliorated by administration of clioquinol, a membrane permeable zinc chelator, to rats prior to exposure to stress, implying that the reduction of synaptic Zn(2+) by clioquinol participates in this amelioration. To pursue the involvement of corticosterone-mediated Zn(2+) signal in the attenuated CA1 LTP by stress, dynamics of synaptic Zn(2+) was checked in hippocampal slices exposed to corticosterone. Corticosterone increased extracellular Zn(2+) levels measured with ZnAF-2 dose-dependently, as well as the intracellular Ca(2+) levels measured with calcium orange AM, suggesting that corticosterone excites zincergic neurons in the hippocampus and increases Zn(2+) release from the neuron terminals. Intracellular Zn(2+) levels measured with ZnAF-2DA were also increased dose-dependently, but not in the coexistence of CaEDTA, a membrane-impermeable zinc chelator, suggesting that intracellular Zn(2+) levels is increased by the influx of extracellular Zn(2+). Furthermore, corticosterone-induced attenuation of CA1 LTP was abolished in the coexistence of CaEDTA. The present study suggests that corticosterone-mediated increase in postsynaptic Zn(2+) signal in the cytosolic compartment is involved in the attenuation of CA1 LTP after exposure to acute stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

PubMed

Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

2014-10-01

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. © 2014 Mello et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

PubMed Central

Kenyon, Amy; Dhanani, Karim C. H.; Prinsloo, Earl; Edkins, Adrienne L.

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis. PMID:24466266

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells.

PubMed

Hunter, Morgan C; O'Hagan, Kyle L; Kenyon, Amy; Dhanani, Karim C H; Prinsloo, Earl; Edkins, Adrienne L

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cip1 expression.

PubMed

Zuckerbraun, Brian S; Shapiro, Richard A; Billiar, Timothy R; Tzeng, Edith

2003-08-19

The 42/44-kD mitogen-activated protein kinases (extracellular signal-regulated kinases, ERKs) regulate smooth muscle cell (SMC) cell-cycle progression and can either promote or inhibit proliferation depending on the activation status of the small GTPase RhoA. RhoA is involved in the regulation of the actin cytoskeleton and converges on multiple signaling pathways. However, the mechanism by which RhoA modulates ERK signaling is not well defined. The purpose of this investigation was to examine whether RhoA regulates ERK downstream signaling and cellular proliferation through its effects on the cytoskeleton and the nuclear localization of ERK. Treatment of SMCs with Clostridia botulinum C3 exoenzyme, which inhibits RhoA activation, decreased SMC proliferation to 24+/-7% of that of controls and increased p21Waf1/Cip1 transcription and protein levels. These effects of RhoA were reversed by inhibition of ERK phosphorylation. However, inactivation of RhoA did not alter levels of ERK phosphorylation but did increase nuclear localization of phosphorylated ERK. In addition, immunostaining demonstrated that phosphorylated ERK associated with the actin cytoskeleton, which was disrupted by C3 exoenzyme. Leptomycin B, an inhibitor of Crm1 that results in ERK nuclear accumulation, similarly increased p21Waf1/Cip1. RhoA inhibition increased levels of phosphorylated ERK in the cell nucleus. Inhibition of RhoA or pharmacological inhibition of nuclear export resulted in increased p21Waf1/Cip1 expression and decreased SMC proliferation, effects that were partially dependent on ERK. RhoA regulation of the actin cytoskeleton may determine ERK subcellular localization and its subsequent effects on SMC proliferation.

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia

PubMed Central

Cao, Muhua; Li, Tao; He, Zhangxiu; Wang, Lixiu; Yang, Xiaoyan; Kou, Yan; Zou, Lili; Dong, Xue; Novakovic, Valerie A.; Bi, Yayan; Kou, Junjie; Yu, Bo; Fang, Shaohong; Wang, Jinghua; Zhou, Jin

2017-01-01

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation. PMID:28053193

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

The intracellular angiotensin system buffers deleterious effects of the extracellular paracrine system

PubMed Central

Villar-Cheda, Begoña; Costa-Besada, Maria A; Valenzuela, Rita; Perez-Costas, Emma; Melendez-Ferro, Miguel; Labandeira-Garcia, Jose L

2017-01-01

The ‘classical’ renin–angiotensin system (RAS) is a circulating system that controls blood pressure. Local/paracrine RAS, identified in a variety of tissues, including the brain, is involved in different functions and diseases, and RAS blockers are commonly used in clinical practice. A third type of RAS (intracellular/intracrine RAS) has been observed in some types of cells, including neurons. However, its role is still unknown. The present results indicate that in brain cells the intracellular RAS counteracts the intracellular superoxide/H2O2 and oxidative stress induced by the extracellular/paracrine angiotensin II acting on plasma membrane receptors. Activation of nuclear receptors by intracellular or internalized angiotensin triggers a number of mechanisms that protect the cell, such as an increase in the levels of protective angiotensin type 2 receptors, intracellular angiotensin, PGC-1α and IGF-1/SIRT1. Interestingly, this protective mechanism is altered in isolated nuclei from brains of aged animals. The present results indicate that at least in the brain, AT1 receptor blockers acting only on the extracellular or paracrine RAS may offer better protection of cells. PMID:28880266

Fungal melanins and their interactions with metals.

PubMed

Fogarty, R V; Tobin, J M

1996-09-01

Fungal melanins are dark brown or black pigments located in cell walls. They also exist as extracellular polymers. Melanized fungi possess increased virulence and resistance to microbial attack as well as enhanced survival while under environmental stress. Melanins contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions. Pigmented Cladosporium cladosporoides was shown to biosorb 2.5- to four-fold more Ni, Cu, Zn, Cd, and Pb than albino Penicillium digitatum and at four- to six-fold higher rates. Metal desorption was significantly lower for extracellular melanin than from pigmented or albino biomass which indicated the strength of the melanin-metal bond. At equilibrium, tributyltin chloride (TBTC) concentrations of 2.5 mM, pigmented and albino Aureobasidium pullulans absorbed approximately 0.9 and 0.7 mumol TBTC mg -1 dry wt, respectively, whereas purified extracellular melanin exhibited uptake levels of approximately 22 mumol TBTC mg-1 dry wt at an equilibrium concentration of only 0.4 mM. Addition of melanin to the growth medium reduced the toxic effect of CuSO4 and TBTC due to melanin metal binding and sequestration.

Effects of amantadine and budipine on antidepressant drug-evoked changes in extracellular 5-HT in the frontal cortex of freely moving rats

PubMed Central

Owen, Jenny C E; Whitton, Peter S

2005-01-01

Evidence has recently suggested that NMDA receptors may play a role in the aetiology and possible treatment of depression and that weak noncompetitive NMDA receptor antagonists such as amantadine can synergize with conventional antidepressants in a model of the illness. To try to obtain a neurochemical rationale for these findings, we have studied the effects of acute and chronic administration of amantadine or the related drug budipine on cortical release of 5-hydroxytryptamine (5-HT) following the antidepressants reboxitine (REB), paroxetine (PAROX) and clomipramine (CLOM) in freely moving rats by using microdialysis. Acute administration of amantadine (40 mg kg−1), budipine (10 mg kg−1), REB (10 mg kg−1), PAROX (10 mg kg−1) or CLOM (10 mg kg−1) all failed to significantly alter extracellular 5-HT in the cortex. However, when either amantadine or budipine was administered 30 min prior to any of the three antidepressants, a significant rise in 5-HT was observed. For chronic studies, the effects of the drugs were studied at 4, 7, 14 and 21 days. Amantadine and budipine did not significantly alter extracellular 5-HT at any time point. The three antidepressant drugs all elicited a gradual increase in 5-HT, which became significant after 14 days and tended to plateau thereafter. When either amantadine (20 mg kg−1) or budipine (5 mg kg−1) was coadministered with any of the three antidepressants, two differences were seen compared with the effects of the antidepressants alone. Firstly, the time required for significant increases in cortical 5-HT was reduced with elevated levels now being observed by 7 days. Secondly, the absolute magnitude of the increase in extracellular 5-HT was markedly greater in these rats from day 7 until the end of the experiment. If, as is widely considered, an increase in extracellular 5-HT represents a critical step in the mechanism of action of antidepressants, these data suggest that combined treatment with clinically tolerated NMDA antagonists such as amantadine could reduce the delay in therapeutic onset of antidepressants as well as possibly enhance their efficacy. PMID:15834446

Changes in Purines Concentration in the Cerebrospinal Fluid of Pregnant Women Experiencing Pain During Active Labor.

PubMed

Schmidt, André P; Böhmer, Ana E; Hansel, Gisele; Soares, Félix A; Oses, Jean P; Giordani, Alex T; Posso, Irimar P; Auler, José Otávio C; Mendes, Florentino F; Félix, Elaine A; Portela, Luís V; Souza, Diogo O

2015-11-01

Labor pain has been reported as a severe pain and can be considered as a model of acute visceral pain. It is well known that extracellular purines have an important role in pain signaling in the central nervous system. This study analyzes the relationship between extracellular purines and pain perception during active labor. A prospective observational study was performed. Cerebrospinal fluid (CSF) levels of the purines and their metabolites were compared between women at term pregnancy with labor pain (n = 49) and without labor pain (Caesarian section; n = 47). Control groups (healthy men and women without chronic or acute pain-n = 40 and 32, respectively) were also investigated. The CSF levels of adenosine were significantly lower in the labor pain group (P = 0.026) and negatively correlated with pain intensity measured by a visual analogue scale (r = -0.48, P = 0.0005). Interestingly, CSF levels of uric acid were significantly higher in healthy men as compared to women. Additionally, pregnant women showed increased CSF levels of ADP, GDP, adenosine and guanosine and reduced CSF levels of AMP, GTP, and uric acid as compared to non-pregnant women (P < 0.05). These findings suggest that purines, in special the nucleoside adenosine, are associated with pregnancy and labor pain.

Detection of Pathological Changes in the Aorta during Thoracic Aortic Aneurysm Progression on Molecular Level

PubMed Central

Rabajdová, Miroslava; Špaková, Ivana; Panagiotis, Artemiou; Ferenčáková, Michaela; Rybár, Dušan; Bobrov, Nikita; Sabol, František

2017-01-01

The progression of thoracic aortic aneurysm depends on regulation of aortic wall homeostasis and on changes in the structural components of the extracellular matrix, which are affected by multiple molecular signalling pathways. We decided to correlate the diameter of ascending thoracic aneurysm with gene expression of inflammation markers (IL-6, CRP), cytokine receptors (IL-6R, TNFR1, and TNFR2), and extracellular matrix components (Emilin-1, MMP9, and TIMP) for detection of the degree of pathological process of TAA formation. The experimental group was divided into three groups according to the diameter of the aortic aneurysm. Whole blood and tissue samples were properly collected and used for nucleic acid, chromatin, and protein isolation. The mRNA levels were detected by qRT-PCR. For the detection of protein levels a Cytokine Array IV assay kit was used in combination with a biochip analyzer. In aortic tissue, significant positive correlations were found between increased mRNA levels of inflammatory cytokines (CRP and IL-6) on both mRNA levels in tissue and protein from the blood with maximum in stage 3. Changes of gene expression of selected genes can be used for the experimental study of the inflammatory receptor inhibitors during trials targeted on slowing down the progress of aortic wall aneurysm. PMID:29158612

Synthesis, maturation and extracellular release of procathepsin D as influenced by cell proliferation or transformation.

PubMed

Isidoro, C; Demoz, M; De Stefanis, D; Baccino, F M; Bonelli, G

1995-12-11

The relationship between cell growth and intra- and extracellular accumulation of cathepsin D (CD), a lysosomal endopeptidase involved in cell protein breakdown, was examined in cultures of normal and transformed BALB/c mouse 3T3 fibroblasts grown at various cell densities. In crowded cultures of normal 3T3 cells (doubling time, Td, 53 hr) intracellular CD activity was 2-fold higher than in sparse, rapidly-growing (Td, 27 hr) cultures. In uncrowded (Td, 18 hr) and crowded (Td, 32 hr) cultures of benzo[a]pyrene-transformed cells intracellular CD levels were one third and two thirds, respectively, of those measured in hyperconfluent 3T3 cultures. Regardless of cell density, SV-40-virus-transformed cells (Td, 12 hr) contained one third of CD levels found in hyperconfluent 3T3 cells. Both transformed cell lines released into the medium a higher proportion of CD, compared with their untransformed counterpart, yet the amount secreted was not sufficient to account for the reduced intracellular level of the enzyme. Serum withdrawal induced a marked increase of both intra- and extracellular levels of CD activity. In both normal and virally or chemically transformed 3T3 cells CD comprised a precursor (52 kDa) and processed mature polypeptides; the latter were mostly represented by a 48-kDa peptide, but a minor part was in a double-chain form (31 and 16 kDa respectively). The proportion of mature enzyme vs. precursor was much higher in confluent, slowly-growing cells than in fast-growing cells, whether normal or transformed. In the latter, conversion of mature 48-kDa peptide into the double-chain form occurred more efficiently.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

PubMed

Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

2012-01-10

The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Modeling extracellular fields for a three-dimensional network of cells using NEURON.

PubMed

Appukuttan, Shailesh; Brain, Keith L; Manchanda, Rohit

2017-10-01

Computational modeling of biological cells usually ignores their extracellular fields, assuming them to be inconsequential. Though such an assumption might be justified in certain cases, it is debatable for networks of tightly packed cells, such as in the central nervous system and the syncytial tissues of cardiac and smooth muscle. In the present work, we demonstrate a technique to couple the extracellular fields of individual cells within the NEURON simulation environment. The existing features of the simulator are extended by explicitly defining current balance equations, resulting in the coupling of the extracellular fields of adjacent cells. With this technique, we achieved continuity of extracellular space for a network model, thereby allowing the exploration of extracellular interactions computationally. Using a three-dimensional network model, passive and active electrical properties were evaluated under varying levels of extracellular volumes. Simultaneous intracellular and extracellular recordings for synaptic and action potentials were analyzed, and the potential of ephaptic transmission towards functional coupling of cells was explored. We have implemented a true bi-domain representation of a network of cells, with the extracellular domain being continuous throughout the entire model. This has hitherto not been achieved using NEURON, or other compartmental modeling platforms. We have demonstrated the coupling of the extracellular field of every cell in a three-dimensional model to obtain a continuous uniform extracellular space. This technique provides a framework for the investigation of interactions in tightly packed networks of cells via their extracellular fields. Copyright © 2017 Elsevier B.V. All rights reserved.

A perspective on extracellular vesicles proteomics

NASA Astrophysics Data System (ADS)

Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

2017-11-01

Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieve from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation.

PubMed

Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J; Pellerin, Luc

2005-11-08

A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

PubMed Central

Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

2015-01-01

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

PubMed

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation

PubMed Central

Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J.; Pellerin, Luc

2005-01-01

A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans. PMID:16260743

Ethanol-induced dopamine elevation in the rat--modulatory effects by subchronic treatment with nicotinic drugs.

PubMed

Löf, Elin; Chau, Pei Pei; Stomberg, Rosita; Söderpalm, Bo

2007-01-26

Chronic nicotine administration is associated with increased ethanol consumption in laboratory animals and in humans. Some smokers report less sedation during acute ethanol intoxication after nicotine administration and the sedative effects from ethanol are mediated by inhibitory GABA(A)-receptors. In a series of in vivo microdialysis experiments we investigated whether subchronic pre-treatment with nicotinic drugs known to enhance ethanol consumption in the rat (nicotine or the peripheral nicotinic antagonist hexamethonium) could modulate the alterations in extracellular dopamine observed in response to administration of ethanol or the sedative GABA(A)-agonist diazepam. In the nucleus accumbens and the dorsal striatum, systemic and/or local ethanol administration resulted in transient increases in extracellular dopamine levels that returned to baseline before the local levels of ethanol started to decline. In hexamethonium pre-treated rats, however, the nucleus accumbens dopamine levels were time-locked to the ethanol levels in the same area after systemic or local ethanol administration. Perfusion of diazepam into the nucleus accumbens produced a significant reduction in nucleus accumbens dopamine in controls. Prior subchronic treatment with nicotine or hexamethonium abolished this effect. The present results suggest that subchronic treatment with the nicotinic acetylcholine receptor antagonist hexamethonium reduces a GABA(A)-R mediated counteraction of the nucleus accumbens dopamine response to ethanol. Additionally, we demonstrate that modulation of nicotinic receptors may reduce the sensitivity of GABA(A) receptors to benzodiazepines. These phenomena may offer a novel explanation to why nicotine and alcohol are often co-abused.

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

PubMed Central

Conley, Jason M.

2017-01-01

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens. PMID:29121644

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

PubMed

Alonso, Carlos Agustín I; Osycka-Salut, Claudia E; Castellano, Luciana; Cesari, Andreína; Di Siervi, Nicolás; Mutto, Adrián; Johannisson, Anders; Morrell, Jane M; Davio, Carlos; Perez-Martinez, Silvina

2017-08-01

Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 μg/ml) and bicarbonate (40 mM). Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). None. This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Astrocytic and neuronal accumulation of elevated extracellular K+ with a 2/3 K+/Na+ flux ratio—consequences for energy metabolism, osmolarity and higher brain function

PubMed Central

Hertz, Leif; Xu, Junnan; Song, Dan; Yan, Enzhi; Gu, Li; Peng, Liang

2013-01-01

Brain excitation increases neuronal Na+ concentration by 2 major mechanisms: (i) Na+ influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na+ influx followed by repolarizating K+ efflux, increasing extracellular K+ concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K+ is initially mainly effectuated by Na+,K+-ATPase-mediated K+ uptake into astrocytes, at K+ concentrations above ~10 mM aided by uptake of Na+,K+ and 2 Cl− by the cotransporter NKCC1. Since operation of the astrocytic Na+,K+-ATPase requires K+-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K+ concentration. This allows K+ release via the inward rectifying K+ channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K+ transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K+ release would have little effect on extracellular K+ concentration and allow K+ accumulation by the less powerful neuronal Na+,K+-ATPase, which is not stimulated by increases in extracellular K+. Since the Na+,K+-ATPase exchanges 3 Na+ with 2 K+, it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by β-adrenergic stimulation of the Na+,K+-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K+]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na+,K+-ATPase fluxes. PMID:23986689

Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion.

PubMed

Syed, Samreen K; Kauffman, Audra L; Beavers, Lisa S; Alston, James T; Farb, Thomas B; Ficorilli, James; Marcelo, Marialuisa C; Brenner, Martin B; Bokvist, Krister; Barrett, David G; Efanov, Alexander M

2013-11-15

Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

Expression of heat shock protein 47 is increased in remnant kidney and correlates with disease progression

PubMed Central

SUNAMOTO, MASAAKI; KUZE, KOGO; IEHARA, NORIYUKI; TAKEOKA, HIROYA; NAGATA, KAZUHIRO; KITA, TORU; DOI, TOSHIO

1998-01-01

Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and IV collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progresive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis. PMID:9741355

[Appropriate usage of antibiotics by therapeutic drug monitoring].

PubMed

Kokubun, Hideya; Kimura, Toshimi; Yago, Kazuo

2007-06-01

Aminoglycosides are mainly distributed in the extracellular fluid, so when they are given to neonates who have a large amount of extracellular fluid, their distribution is increased. In our data, the volume of distribution (Vd) of Arbekacin in the neonates was twice that of the adults, 0.54 l/kg. Therefore, the dose per weight of aminoglycosides to the neonates should be increased more than to the adults. In the renal function of the neonates, differentiation of the nephron is completed within 36 weeks after conception, but it is functionally immature. In our data, renal drug excretion increased rapidly in the post-conceptional ages (PCAs) of 34-35 weeks. Consequently, we based the Arbekacin administration schedule for the neonates on the PCAs. There is excellent correlation between serum level of vancomicin (VCM) and dose x serum creatinine (Scr)/weight in the haemodialysis patients, suggesting that we can use weight and Scr to set the VCM administration schedule for these patients. We also established on administration schedule of Teicoplanin for the haemodialysis patients. In this article, we present the TDM analysis result of the antibiotics in our hospital.

Extracellular growth factors and mitogens cooperate to drive mitochondrial biogenesis

PubMed Central

Echave, Pedro; Machado-da-Silva, Gisela; Arkell, Rebecca S.; Duchen, Michael R.; Jacobson, Jake; Mitter, Richard; Lloyd, Alison C.

2009-01-01

Summary Cells generate new organelles when stimulated by extracellular factors to grow and divide; however, little is known about how growth and mitogenic signalling pathways regulate organelle biogenesis. Using mitochondria as a model organelle, we have investigated this problem in primary Schwann cells, for which distinct factors act solely as mitogens (neuregulin) or as promoters of cell growth (insulin-like growth factor 1; IGF1). We find that neuregulin and IGF1 act synergistically to increase mitochondrial biogenesis and mitochondrial DNA replication, resulting in increased mitochondrial density in these cells. Moreover, constitutive oncogenic Ras signalling results in a further increase in mitochondrial density. This synergistic effect is seen at the global transcriptional level, requires both the ERK and phosphoinositide 3-kinase (PI3K) signalling pathways and is mediated by the transcription factor ERRα. Interestingly, the effect is independent of Akt-TOR signalling, a major regulator of cell growth in these cells. This separation of the pathways that drive mitochondrial biogenesis and cell growth provides a mechanism for the modulation of mitochondrial density according to the metabolic requirements of the cell. PMID:19920079

Involvement of serotonin in the ventral tegmental area in thermoregulation of freely moving rats.

PubMed

Ishiwata, Takayuki; Hasegawa, Hiroshi; Greenwood, Benjamin N

2017-07-13

We have recently reported that the serotonin (5-HT) projections from the midbrain's raphe nuclei that contains 5-HT cell bodies may play a role both in heat production and in heat loss. The purpose of the present study was to clarify the involvement of 5-HT in the ventral tegmental area (VTA), where 5-HT is suggested to participate in thermoregulation, using the combined methods of telemetry, microdialysis, and high performance liquid chromatography, with a special emphasis on regulation of the body temperature (T b ) in freely moving rats. First, we measured changes in T b , tail skin temperature (T tail ; an index of heat loss), heart rate (HR; an index of heat production), locomotor activity (Act), and levels of extracellular monoamines in the VTA during cold (5°C) or heat (35°C) exposure. Subsequently, we perfused citalopram (5-HT re-uptake inhibitor) into the VTA and measured the thermoregulatory parameters and monoamines release. Although T b , T tail , and HR changed during both exposures, significant changes in extracellular level of 5-HT (138.7±12.7% baseline, p<0.01), but not dopamine (DA) or noradrenaline (NA) were noted in the VTA only during heat exposure. In addition, perfusion of citalopram into the VTA increased extracellular 5-HT levels (221.0±52.2% baseline, p<0.01), but not DA or NA, while T b decreased from 37.4±0.1°C to 36.8±0.2°C (p<0.001),T tail increased from 26.3±0.4°C to 28.4±0.4°C (p<0.001), and HR and Act remained unchanged. Our results suggest that the VTA is a key area for thermoregulation, and 5-HT, but not DA or NA, modulates the heat loss system through action in the VTA. Copyright © 2017 Elsevier B.V. All rights reserved.

Co-Administration of Ethanol and Nicotine: The Enduring Alterations in the Rewarding Properties of Nicotine and Glutamate Activity within the Mesocorticolimbic System of Female Alcohol-Preferring (P) Rats

PubMed Central

Deehan, Gerald A.; Hauser, Sheketha R.; Waeiss, R. Aaron; Knight, Christopher P.; Toalston, Jamie E.; Truitt, William A.; McBride, William J.; Rodd, Zachary A.

2016-01-01

Rationale The co-abuse of ethanol (EtOH) and nicotine (NIC) increases the likelihood that an individual will relapse to drug-use while attempting to maintain abstinence. There is limited research examining the consequences of long-term EtOH and NIC co-abuse. Objectives The current experiments determined the enduring effects of chronic EtOH, NIC, or EtOH + NIC intake on the reinforcing properties of NIC and glutamate (GLU) activity within the mesocorticolimbic (MCL) system. Methods Alcohol-preferring (P) rats self-administered EtOH, Sacc + NIC or EtOH + NIC combined for 10 weeks. The reinforcing properties of 0.1–3.0 uM NIC within the nucleus accumbens shell (AcbSh) were assessed following a 2–3 week drug-free period using intracranial self-administration (ICSA) procedures. The effects of EtOH, Sacc, Sacc + NIC or EtOH + NIC intake on extracellular levels and clearance of glutamate (GLU) in the medial prefrontal cortex (mPFC) were also determined. Results Binge intake of EtOH (96–100 mg%) and NIC (21–27 mg/ml) were attained. All groups of P rats self-infused 3.0 uM NIC directly into the AcbSh; whereas only animals in the EtOH + NIC co-abuse group self-infused the 0.3 and 1.0 uM NIC concentrations. Additionally, self-administration of EtOH + NIC, but not EtOH, Sacc or Sacc + NIC, resulted in enduring increases in basal extracellular GLU levels in the mPFC. Conclusions Overall, the co-abuse of EtOH + NIC produced enduring neuronal alterations within the MCL which enhanced the rewarding properties of NIC in the AcbSh and elevated extracellular GLU levels within the mPFC. PMID:26306917

Exosome release and low pH belong to a framework of resistance of human melanoma cells to cisplatin.

PubMed

Federici, Cristina; Petrucci, Francesco; Caimi, Stefano; Cesolini, Albino; Logozzi, Mariantonia; Borghi, Martina; D'Ilio, Sonia; Lugini, Luana; Violante, Nicola; Azzarito, Tommaso; Majorani, Costanza; Brambilla, Daria; Fais, Stefano

2014-01-01

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin

PubMed Central

Federici, Cristina; Petrucci, Francesco; Caimi, Stefano; Cesolini, Albino; Logozzi, Mariantonia; Borghi, Martina; D'Ilio, Sonia; Lugini, Luana; Violante, Nicola; Azzarito, Tommaso; Majorani, Costanza; Brambilla, Daria; Fais, Stefano

2014-01-01

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity. PMID:24516610

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

PubMed

Crosas-Molist, Eva; Meirelles, Thayna; López-Luque, Judit; Serra-Peinado, Carla; Selva, Javier; Caja, Laia; Gorbenko Del Blanco, Darya; Uriarte, Juan José; Bertran, Esther; Mendizábal, Yolanda; Hernández, Vanessa; García-Calero, Carolina; Busnadiego, Oscar; Condom, Enric; Toral, David; Castellà, Manel; Forteza, Alberto; Navajas, Daniel; Sarri, Elisabet; Rodríguez-Pascual, Fernando; Dietz, Harry C; Fabregat, Isabel; Egea, Gustavo

2015-04-01

Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation. © 2015 American Heart Association, Inc.

Plasma Levels of Matrix Metalloproteinase (MMP)-2, MMP-9 and Tumor Necrosis Factor-α in Chronic Hepatitis C Virus Patients.

PubMed

Abdel-Latif, Mohamed S

2015-01-01

In chronic HCV infection, pathological accumulation of the extracellular matrix is the main feature of liver fibrosis; that indicates the imbalanced rate of increased matrix synthesis to decreased breakdown of connective tissue proteins. Matrix metalloproteinases (MMPs) play a crucial role in remodeling of extracellular matrix. It is known that expression of MMPs is regulated by Tumor necrosis factor (TNF)-α. Also, levels of TNF-α in liver and serum are increased in chronic HCV patient. Accordingly, this study aimed to correlate the plasma levels of MMP-2, MMP-9 and TNF-α in chronic HCV patients with the pathogenesis of the liver. The current study was conducted on 15 fibrotic liver cases with detectable HCV RNA, 10 HCV cirrhotic liver cases, and 15 control subjects of matched age and sex. Plasma MMP-2, MMP-9 and TNF-α were measured by ELISA. Data revealed that the MMP2, MMP9 and TNF-α levels showed a significant elevation in chronic HCV patients compared to control group (p= 0.001). But, no significant correlation was observed in levels of MMP-2, MMP-9, and TNF-α between fibrotic and cirrhotic cases. MMP-2, MMP-9 and TNF-α showed high reproducibility to differentiate chronic HCV patients from control group. On the contrary, MMP-2, MMP-9 and TNF-α were not able to differentiate fibrotic from cirrhotic liver cases. Thus, MMP-2, MMP-9 and TNF-α could not be correlated with the progression of liver disease. Rather they could be used as prognostic markers of liver fibrosis.

Social dynamics within decomposer communities lead to nitrogen retention and organic matter build-up in soils

PubMed Central

Kaiser, Christina; Franklin, Oskar; Richter, Andreas; Dieckmann, Ulf

2015-01-01

The chemical structure of organic matter has been shown to be only marginally important for its decomposability by microorganisms. The question of why organic matter does accumulate in the face of powerful microbial degraders is thus key for understanding terrestrial carbon and nitrogen cycling. Here we demonstrate, based on an individual-based microbial community model, that social dynamics among microbes producing extracellular enzymes (‘decomposers') and microbes exploiting the catalytic activities of others (‘cheaters') regulate organic matter turnover. We show that the presence of cheaters increases nitrogen retention and organic matter build-up by downregulating the ratio of extracellular enzymes to total microbial biomass, allowing nitrogen-rich microbial necromass to accumulate. Moreover, increasing catalytic efficiencies of enzymes are outbalanced by a strong negative feedback on enzyme producers, leading to less enzymes being produced at the community level. Our results thus reveal a possible control mechanism that may buffer soil CO2 emissions in a future climate. PMID:26621582

Development of a Sensitive Luciferase-Based Sandwich ELISA System for the Detection of Human Extracellular Matrix 1 Protein.

PubMed

Li, Ya; Li, Yanqing; Zhao, Junli; Zheng, Xiaojing; Mao, Qinwen; Xia, Haibin

2016-12-01

Enzyme-linked immunosorbent assay (ELISA) has been one of the main methods for detecting an antigen in an aqueous sample for more than four decades. Nowadays, one of the biggest concerns for ELISA is still how to improve the sensitivity of the assay, and the luciferase-luciferin reaction system has been noticed as a new detection method with high sensitivity. In this study, a luciferin-luciferase reaction system was used as the detection method for a sandwich ELISA system. It was shown that this new system led to an increase in the detection sensitivity of at least two times when compared with the traditional horseradish peroxidase (HRP) detection method. Lastly, the serum levels of the human extracellular matrix 1 protein of breast cancer patients were determined by the new system, which were overall similar to the HRP chemiluminescent system. Furthermore, this new luciferase reporter can be implemented into other ELISA systems for the purpose of increasing the assay sensitivity.

The essential oil of bergamot enhances the levels of amino acid neurotransmitters in the hippocampus of rat: implication of monoterpene hydrocarbons.

PubMed

Morrone, Luigi A; Rombolà, Laura; Pelle, Cinzia; Corasaniti, Maria T; Zappettini, Simona; Paudice, Paolo; Bonanno, Giambattista; Bagetta, Giacinto

2007-04-01

The effects of bergamot essential oil (BEO) on the release of amino acid neurotransmitters in rat hippocampus have been studied by in vivo microdialysis and by in vitro superfusion of isolated nerve terminals. Intraperitoneal administration of BEO (100microl/kg) significantly elevated the extracellular concentration of aspartate, glycine and taurine in a Ca(2+)-dependent manner. A dose-relation study generated a bell-shaped curve. When perfused into the hippocampus via the dialysis probe (20microl/20min), BEO produced a significant increase of extracellular aspartate, glycine, taurine as well as of GABA and glutamate. The augmentation of all amino acids was Ca(2+)-independent. Focally injected 1:1 diluted BEO preferentially caused extracellular increase of glutamate. Interestingly, this release appeared to be strictly Ca(2+)-dependent. BEO concentration-dependently enhanced the release of [(3)H]D-aspartate from superfused hippocampal synaptosomes. Similar results were obtained by monitoring the BEO-evoked release of endogenous glutamate. At relatively high concentrations, the BEO-induced [(3)H]d-aspartate release was almost entirely prevented by the glutamate transporter blocker dl-threo-beta-benzyloxyaspartic acid (DL-TBOA) and was Ca(2+)-independent. At relatively low concentrations the release of [(3)H]D-aspartate was only in part ( approximately 50%) DL-TBOA-sensitive and Ca(2+)-independent; the remaining portion of release was dependent on extracellular Ca(2+). Interestingly, the monoterpene hydrocarbon-free fraction of the essential oil appeared to be inactive while the bergapten-free fraction superimposed the releasing effect of BEO supporting the deduction that psoralens may not be implicated. To conclude, BEO contains into its volatile fraction still unidentified monoterpene hydrocarbons able to stimulate glutamate release by transporter reversal and/or by exocytosis, depending on the dose administered.

Extracellular superoxide dismutase is necessary to maintain renal blood flow during sepsis development.

PubMed

Constantino, Larissa; Galant, Letícia Selinger; Vuolo, Francieli; Guarido, Karla Lorena; Kist, Luiza Wilges; de Oliveira, Giovanna Medeiros Tavares; Pasquali, Matheus Augusto de Bittencourt; de Souza, Cláudio Teodoro; da Silva-Santos, José Eduardo; Bogo, Maurício Reis; Moreira, José Cláudio Fonseca; Ritter, Cristiane; Dal-Pizzol, Felipe

2017-12-01

Extracellular superoxide dismutase (ECSOD) protects nitric oxide (NO) bioavailability by decreasing superoxide levels and preventing peroxynitrite generation, which is important in maintaining renal blood flow and in preventing acute kidney injury. However, the profile of ECSOD expression after sepsis is not fully understood. Therefore, we intended to evaluate the content and gene expression of superoxide dismutase (SOD) isoforms in the renal artery and their relation to renal blood flow. Sepsis was induced in Wistar rats by caecal ligation and perforation. Several times after sepsis induction, renal blood flow (12, 24 and 48 h); the renal arterial content of SOD isoforms, nitrotyrosine, endothelial and inducible nitric oxide synthase (e-NOS and i-NOS), and phosphorylated vasodilator-stimulated phosphoprotein (pVASP); and SOD activity (3, 6 and 12 h) were measured. The influence of a SOD inhibitor was also evaluated. An increase in ECSOD content was associated with decreased 3-nitrotyrosine levels. These events were associated with an increase in pVASP content and maintenance of renal blood flow. Moreover, previous treatment with a SOD inhibitor increased nitrotyrosine content and reduced renal blood flow. ECSOD appears to have a major role in decreasing peroxynitrite formation in the renal artery during the early stages of sepsis development, and its application can be important in renal blood flow control and maintenance during septic insult.

Intracellular Phosphate Dynamics in Muscle Measured by Magnetic Resonance Spectroscopy during Hemodialysis

PubMed Central

Fournier, Thomas; Kocevar, Gabriel; Belloi, Amélie; Normand, Gabrielle; Ibarrola, Danielle; Sappey-Marinier, Dominique; Juillard, Laurent

2016-01-01

Of the 600–700 mg inorganic phosphate (Pi) removed during a 4-hour hemodialysis session, a maximum of 10% may be extracted from the extracellular space. The origin of the other 90% of removed phosphate is unknown. This study tested the hypothesis that the main source of phosphate removed during hemodialysis is the intracellular compartment. Six binephrectomized pigs each underwent one 3-hour hemodialysis session, during which the extracorporeal circulation blood flow was maintained between 100 and 150 ml/min. To determine in vivo phosphate metabolism, we performed phosphorous (31P) magnetic resonance spectroscopy using a 1.5-Tesla system and a surface coil placed over the gluteal muscle region. 31P magnetic resonance spectra (repetition time =10 s; echo time =0.35 ms) were acquired every 160 seconds before, during, and after dialysis. During the dialysis sessions, plasma phosphate concentrations decreased rapidly (−30.4 %; P=0.003) and then, plateaued before increasing approximately 30 minutes before the end of the sessions; 16 mmol phosphate was removed in each session. When extracellular phosphate levels plateaued, intracellular Pi content increased significantly (11%; P<0.001). Moreover, βATP decreased significantly (P<0.001); however, calcium levels remained balanced. Results of this study show that intracellular Pi is the source of Pi removed during dialysis. The intracellular Pi increase may reflect cellular stress induced by hemodialysis and/or strong intracellular phosphate regulation. PMID:26561642

Cationic composition and acid-base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis.

PubMed

Pirow, Ralph; Buchen, Ina; Richter, Marc; Allmer, Carsten; Nunes, Frank; Günsel, Andreas; Heikens, Wiebke; Lamkemeyer, Tobias; von Reumont, Björn M; Hetz, Stefan K

2009-04-01

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.

Heterogeneity in extracellular nucleotide hydrolysis among clinical isolates of Trichomonas vaginalis

PubMed Central

TASCA, T.; BONAN, C. D.; DE CARLI, G. A.; SARKIS, J.J.F.; ALDERETE, J. F.

2007-01-01

SUMMARY Trichomonas vaginalis is a parasitic protozoan that causes trichomonosis, a sexually-transmitted disease, with serious sequelae to women and men. As the host-parasite relationship is complex, it is important to investigate biochemical aspects of the parasite that contribute to our understanding of trichomonal biology and pathogenesis. Nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1), which hydrolyses extracellular ATP and ADP, and ecto-5′-nucleotidase, which hyrolyses AMP, have been characterized in laboratory isolates of T. vaginalis. Here we show that the extracellular ATP:ADP hydrolysis ratio varies among fresh clinical isolates, which presented higher ATPase and ADPase activities than long-term-grown isolates. Growth of parasites in iron-replete and iron-depleted medium resulted in different, albeit minor, patterns in extracellular ATP and ADP hydrolysis among isolates. Importantly, some isolates had low or absent ecto-5′-nucleotidase activity, regardless of environmental conditions tested. For isolates with ecto-5′-nucleotidase activity, high- and low-iron trichomonads had increased and decreased levels of activity, respectively, compared to organisms grown in normal TYM-serum medium. This suggests a regulation in expression of either the enzyme amounts and/or activity under the control of iron. Finally, we found no correlation between the presence or absence of dsRNA virus infection among trichomonad isolates and NTPDase and ecto-5′-nucleotidase activities. PMID:16038398

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

PubMed

Schrattenholz, A; Roth, U; Godovac-Zimmermann, J; Maelicke, A

1997-10-28

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Role of CNPase in the Oligodendrocytic Extracellular 2′,3′-cAMP-Adenosine Pathway

PubMed Central

Verrier, Jonathan D.; Jackson, Travis C.; Gillespie, Delbert G.; Janesko-Feldman, Keri; Bansal, Rashmi; Goebbels, Sandra; Nave, Klaus-Armin; Kochanek, Patrick M.; Jackson, Edwin K.

2014-01-01

Extracellular adenosine 3′,5′-cyclic monophosphate (3′,5′-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′-cAMP to adenosine. Here we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′-cAMP and their respective adenosine monophosphates (2′-AMP and 3′-AMP). Cells were also isolated from mice deficient in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′-cAMP to 2′-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′-AMP was minimal in both oligodendrocytes and neurons. The production of 2′-AMP from 2′,3′-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3′,5′-cAMP-3′-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2′,3′-cAMP to 2′-AMP and inhibition of classic ecto-5′-nucleotidase (CD73) with α,β-methylene-adenosine-5′-diphosphate did not attenuate the conversion of 2′-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′-cAMP to 2-AMP in CNS cells. By reducing levels of 2′,3′-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. PMID:23922219

Filtration recovery of extracellular DNA from environmental water samples

EPA Science Inventory

qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular ...

The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing.

PubMed

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla A; Diekwisch, Tom; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBN(Δ5-6) truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

The Ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing

PubMed Central

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla; Diekwisch, Thomas G.H.; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNΔ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. PMID:26899203

Desialylation of glycoconjugates on the surface of monocytes activates the extracellular signal-related kinases ERK 1/2 and results in enhanced production of specific cytokines.

PubMed

Stamatos, Nicholas M; Curreli, Sabrina; Zella, Davide; Cross, Alan S

2004-02-01

Modulation of the sialic acid content of cell-surface glycoproteins and glycolipids influences the functional capacity of cells of the immune system. The role of sialidase(s) and the consequent desialylation of cell surface glycoconjugates in the activation of monocytes have not been established. In this study, we show that desialylation of glycoconjugates on the surface of purified monocytes using exogenous neuraminidase (NANase) activated extracellular signal-regulated kinase 1/2 (ERK 1/2), an intermediate in intracellular signaling pathways. Elevated levels of phosphorylated ERK 1/2 were detected in desialylated monocytes after 2 h of NANase treatment, and increased amounts persisted for at least 2 additional hours. Desialylation of cell surface glycoconjugates also led to increased production of interleukin (IL)-6, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by NANase-treated monocytes that were maintained in culture. Neither increased levels of phosphorylated ERK 1/2 nor enhanced production of cytokines were detected when NANase was heat-inactivated before use, demonstrating the specificity of NANase action. Treatment of monocytes with gram-negative bacterial lipopolysaccharide (LPS) also led to enhanced production of IL-6, MIP-1alpha, and MIP-1beta. The amount of each of these cytokines that was produced was markedly increased when monocytes were desialylated with NANase before exposure to LPS. These results suggest that changes in the sialic acid content of surface glycoconjugates influence the activation of monocytes.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latham, Antony M.; Odell, Adam F.; Mughal, Nadeem A.

2012-11-01

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a highmore » VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black-Right-Pointing-Pointer Endothelial VEGFR levels are modulated during this response. Black-Right-Pointing-Pointer The cell regulates VEGF-A bioavailability and cell survival. Black-Right-Pointing-Pointer This may partly underlie endothelial dysfunction seen in many pathologies.« less

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

USGS Publications Warehouse

Redman, Regina S.; Rodriguez, Rusty J.

2002-01-01

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

USGS Publications Warehouse

Redman, R.S.; Rodriguez, R.J.

2002-01-01

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes, causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a uv-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes, and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

PubMed Central

Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

2015-01-01

Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

PubMed

Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

2016-01-01

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.

Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

PubMed

Levay, Agata K; Peacock, Jacqueline D; Lu, Yinhui; Koch, Manuel; Hinton, Robert B; Kadler, Karl E; Lincoln, Joy

2008-10-24

Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

The oxidative stress-inducible cystine/glutamate antiporter, system x (c) (-) : cystine supplier and beyond.

PubMed

Conrad, Marcus; Sato, Hideyo

2012-01-01

The oxidative stress-inducible cystine/glutamate exchange system, system x (c) (-) , transports one molecule of cystine, the oxidized form of cysteine, into cells and thereby releases one molecule of glutamate into the extracellular space. It consists of two protein components, the 4F2 heavy chain, necessary for membrane location of the heterodimer, and the xCT protein, responsible for transport activity. Previously, system x (c) (-) has been regarded to be a mere supplier of cysteine to cells for the synthesis of proteins and the antioxidant glutathione (GSH). In that sense, oxygen, electrophilic agents, and bacterial lipopolysaccharide trigger xCT expression to accommodate with increased oxidative stress by stimulating GSH biosynthesis. However, emerging evidence established that system x (c) (-) may act on its own as a GSH-independent redox system by sustaining a redox cycle over the plasma membrane. Hallmarks of this cycle are cystine uptake, intracellular reduction to cysteine and secretion of the surplus of cysteine into the extracellular space. Consequently, increased levels of extracellular cysteine provide a reducing microenvironment required for proper cell signaling and communication, e.g. as already shown for the mechanism of T cell activation. By contrast, the enhanced release of glutamate in exchange with cystine may trigger neurodegeneration due to glutamate-induced cytotoxic processes. This review aims to provide a comprehensive picture from the early days of system x (c) (-) research up to now.

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

PubMed

Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

2015-07-01

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays.

PubMed

Ohshima, Yasuhiro; Kitami, Akihiro; Kawano, Ayumi; Tsukimoto, Mitsutoshi; Kojima, Shuji

2011-09-15

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cervia, Davide, E-mail: d.cervia@unitus.it; Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano; Catalani, Elisabetta

Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected.more » Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.« less

Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture.

PubMed Central

Pavenstädt, H.; Gloy, J.; Leipziger, J.; Klär, B.; Pfeilschifter, J.; Schollmeyer, P.; Greger, R.

1993-01-01

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 PMID:7691366

Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes

PubMed Central

Sawaki, Daigo; Hou, Lianguo; Tomida, Shota; Sun, Junqing; Zhan, Hong; Aizawa, Kenichi; Son, Bo-Kyung; Kariya, Taro; Takimoto, Eiki; Otsu, Kinya; Conway, Simon J.; Manabe, Ichiro; Komuro, Issei; Friedman, Scott L.; Nagai, Ryozo; Suzuki, Toru

2015-01-01

Aims Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. Methods and results Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. Conclusion Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts. PMID:25987545

Inhibitory effect of fluvoxamine on β-casein expression via a serotonin-independent mechanism in human mammary epithelial cells.

PubMed

Chiba, Takeshi; Maeda, Tomoji; Kimura, Soichiro; Morimoto, Yasunori; Sanbe, Atsushi; Ueda, Hideo; Kudo, Kenzo

2015-11-05

Selective serotonin reuptake inhibitors (SSRIs) are widely used as a first-line therapy in postpartum depression. The objective of this study was to determine the mechanism underlying the inhibitory effects of the SSRI, fluvoxamine, on β-casein expression, an indicator of lactation, in MCF-12A human mammary epithelial cells. Expression levels of serotonin (5-hydroxytryptamine; 5-HT) transporter, an SSRI target protein, and tryptophan hydroxylase 1, a rate-limiting enzyme in 5-HT biosynthesis, were increased in MCF-12A cells by prolactin treatment. Treatment with 1 μM fluvoxamine for 72 h significantly decreased protein levels of β-casein and phosphorylated signal transducer and activator transcription 5 (pSTAT5). Extracellular 5-HT levels were significantly increased after exposure to 1 μM fluvoxamine, in comparison with those of untreated and vehicle-treated cells; however, extracellular 5-HT had little effect on the decrease in β-casein expression. Expression of glucose-related protein 78/binding immunoglobulin protein, a regulator of endoplasmic reticulum (ER) stress, was significantly increased after treatment with 1 μM fluvoxamine for 48 h. Exposure to tunicamycin, an inducer of ER stress, also decreased expression of β-casein and pSTAT5 in a manner similar to fluvoxamine. Our results indicate that fluvoxamine suppresses β-casein expression in MCF-12A cells via inhibition of STAT5 phosphorylation caused by induction of ER stress. Further studies are required to confirm the effect of fluvoxamine on the function of mammary epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression.

PubMed

Zheng, Yuxuan; Ritzenthaler, Jeffrey D; Burke, Tom J; Otero, Javier; Roman, Jesse; Watson, Walter H

2018-04-01

Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain a more oxidizing extracellular redox potential (E h (Cys/CySS)) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular E h (Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and E h (Cys/CySS) was calculated from the Nernst equation. The results showed that both E h (Cys/CySS) and E h (GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular E h (Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing E h (Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore E h (GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study reveals Slc7a11 is the key regulator of age-dependent changes in extracellular E h (Cys/CySS) in primary mouse lung fibroblasts, and its effects are not dependent on GSH synthesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging

PubMed Central

Feng, Juan; Navratil, Marian; Thompson, LaDora V.

2011-01-01

The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present. PMID:18548236

L-Tyrosine availability affects basal and stimulated catecholamine indices in prefrontal cortex and striatum of the rat.

PubMed

Brodnik, Zachary D; Double, Manda; España, Rodrigo A; Jaskiw, George E

2017-09-01

We previously found that L-tyrosine (L-TYR) but not D-TYR administered by reverse dialysis elevated catecholamine synthesis in vivo in medial prefrontal cortex (MPFC) and striatum of the rat (Brodnik et al., 2012). We now report L-TYR effects on extracellular levels of catecholamines and their metabolites. In MPFC, reverse dialysis of L-TYR elevated in vivo levels of dihydroxyphenylacetic acid (DOPAC) (L-TYR 250-1000 μM), homovanillic acid (HVA) (L-TYR 1000 μM) and 3-methoxy-4-hydroxyphenylglycol (MHPG) (L-TYR 500-1000 μM). In striatum L-TYR 250 μM elevated DOPAC. We also examined L-TYR effects on extracellular dopamine (DA) and norepinephrine (NE) levels during two 30 min pulses (P2 and P1) of K+ (37.5 mM) separated by t = 2.0 h. L-TYR significantly elevated the ratio P2/P1 for DA (L-TYR 125 μM) and NE (L-TYR 125-250 μM) in MPFC but lowered P2/P1 for DA (L-TYR 250 μM) in striatum. Finally, we measured DA levels in brain slices using ex-vivo voltammetry. Perfusion with L-TYR (12.5-50 μM) dose-dependently elevated stimulated DA levels in striatum. In all the above studies, D-TYR had no effect. We conclude that acute increases within the physiological range of L-TYR levels can increase catecholamine metabolism and efflux in MPFC and striatum. Chronically, such repeated increases in L-TYR availability could induce adaptive changes in catecholamine transmission while amplifying the metabolic cost of catecholamine synthesis and degradation. This has implications for neuropsychiatric conditions in which neurotoxicity and/or disordered L-TYR transport have been implicated. Published by Elsevier Ltd.

Cellulose effects on morphology and elasticity of Vibrio fischeri biofilms.

PubMed

Ziemba, Christopher; Shabtai, Yael; Piatkovsky, Maria; Herzberg, Moshe

2016-01-01

Cellulose effects on Vibrio fischeri biofilm morphology were tested for the wild-type and two of its isogenic mutants that either exhibit increased cellulose production or do not produce cellulose at all. Confocal laser scanning microscopy imaging of each biofilm revealed that total sessile volume increases with cellulose expression, but the size of colonies formed with cellulose was smaller, creating a more diffuse biofilm. These morphological differences were not attributed to variations in bacterial deposition, extracellular polymeric substances affinity to the surface or bacterial growth. A positive correlation was found between cellulose expression, Young's (elastic) modulus of the biofilm analyzed with atomic force microscope and shear modulus of the related extracellular polymeric substances layers analyzed with quartz crystal microbalance with dissipation monitoring. Cellulose production also correlated positively with concentrations of extracellular DNA. A significant negative correlation was observed between cellulose expression and rates of diffusion through the extracellular polymeric substances. The difference observed in biofilm morphology is suggested as a combined result of cellulose and likely extracellular DNA (i) increasing biofilm Young's modulus, making shear removal more difficult, and (ii) decreased diffusion rate of nutrients and wastes into and out of the biofilm, which effectively limits colony size.

Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

PubMed Central

Besson-Bard, Angélique; Griveau, Sophie; Bedioui, Fethi; Wendehenne, David

2008-01-01

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses. PMID:18653691

Comparative community-level associations of helminth infections and microparasite shedding in wild long-tailed macaques in Bali, Indonesia.

PubMed

Wilcox, Justin J S; Lane-Degraaf, Kelly E; Fuentes, Agustin; Hollocher, Hope

2015-03-01

Helminthes have the capacity to modulate host immunity, leading to positive interactions with coinfecting microparasites. This phenomenon has been primarily studied during coinfections with a narrow range of geo-helminthes and intracellular microparasites in human populations or under laboratory conditions. Far less is known regarding differences in coinfection dynamics between helminth types, the range of microparasites that might be affected or the overall community-level effects of helminth infections on microparasites in wild systems. Here, we analysed the presence/absence and abundance patterns of enteric parasites in long-tailed macaques (Macaca fascicularis) on the island of Bali, Indonesia, to assess whether naturally occurring helminth infections were associated with increased shedding of the most common intracellular (Cryptosporidium spp., Isospora spp.) and extracellular (Entamoeba spp., Giardia spp.) microparasites. We also comparatively assessed the statistical correlations of different helminth taxa with microparasite shedding to determine if there were consistent relationships between the specific helminth taxa and microparasites. Helminth infections were associated with increased shedding of both intracellular and extracellular microparasites. Platyhelminthes repeatedly displayed strong positive correlations with several microparasites; while nematodes did not. Our results indicate that helminthes can influence microparasite community shedding dynamics under wild conditions, but that trends may be driven by a narrow range of helminthes.

Induced Expression of Rnd3 Is Associated with Transformation of Polarized Epithelial Cells by the Raf–MEK–Extracellular Signal-Regulated Kinase Pathway†

PubMed Central

Hansen, Steen H.; Zegers, Mirjam M. P.; Woodrow, Melissa; Rodriguez-Viciana, Pablo; Chardin, Pierre; Mostov, Keith E.; McMahon, Martin

2000-01-01

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf–MEK–extracellular signal-regulated kinase pathway in epithelial oncogenesis. PMID:11094087

Analysis of extracellular proteins of Aspergillus oryzae grown on soy sauce koji.

PubMed

Liang, Yanchang; Pan, Li; Lin, Ying

2009-01-01

Aspergillus oryzae AS 3.951 is widely used in Chinese soy sauce manufacture, but little is known about the profiles of the extracellular proteins from the culture of soybean koji. In this study, we carried out MALDI-TOF/TOF MS analysis of extracellular proteins during koji culture. Besides well-known proteins (TAA and Oryzin), a variety of aminopeptidase and proteases were identical at the proteome level. This suggests that A. oryzae AS 3.951 has a powerful capacity to digest soybean protein.

Classical conditioning leads to changes in extracellular concentrations of ependymin in goldfish brain.

PubMed

Shashoua, V E; Hesse, G W

1989-04-10

ELISA measurements showed that brain extracellular fluid (ECF) levels of ependymin decreased for animals that learned to associate a paired presentation of a light stimulus (CS) with the onset of an electric shock (US), whereas no changes were obtained for control goldfish that received the same number of stimuli delivered in a random unpaired order. Studies of the time course of the changes showed an immediate decrease (19%) after training followed by an increase (20%) above baseline by 5 h and a final return to baseline by 25 h. These data extend the findings of previous experiments, which demonstrated a role for ependymin in two training procedures that involved motor learning, to classical conditioning where no motor learning occurs. Thus it appears that ependymin may have a functional role in molecular mechanisms of learning and memory in general.

Kalirin and CHD7: novel endothelial dysfunction indicators in circulating extracellular vesicles from hypertensive patients with albuminuria

PubMed Central

de la Cuesta, Fernando; Baldan-Martin, Montserrat; Moreno-Luna, Rafael; Alvarez-Llamas, Gloria; Gonzalez-Calero, Laura; Mourino-Alvarez, Laura; Sastre-Oliva, Tamara; López, Juan A.; Vázquez, Jesús; Ruiz-Hurtado, Gema; Segura, Julian; Vivanco, Fernando; Ruilope, Luis M.; Barderas, Maria G.

2017-01-01

Despite of the great advances in anti-hypertensive therapies, many patients under Renin-Angiotensin- System (RAS) suppression develop albuminuria, which is a clear indicator of therapeutic inefficiency. Hence, indicators of vascular function are needed to assess patients’ condition and help deciding future therapies. Proteomic analysis of circulating extracellular vesicles (EVs) showed two proteins, kalirin and chromodomain-helicase-DNA-binding protein 7 (CHD7), increased in albuminuric patients. A positive correlation of both with the expression of the endothelial activation marker E-selectin was found in EVs. In vitro analysis using TNFα-treated adult human endothelial cells proved their involvement in endothelial cell activation. Hence, we propose protein levels of kalirin and CHD7 in circulating EVs as novel endothelial dysfunction markers to monitor vascular condition in hypertensive patients with albuminuria. PMID:28152519

Impact of extracellularity on the evolutionary rate of mammalian proteins.

PubMed

Liao, Ben-Yang; Weng, Meng-Pin; Zhang, Jianzhi

2010-01-06

It is of fundamental importance to understand the determinants of the rate of protein evolution. Eukaryotic extracellular proteins are known to evolve faster than intracellular proteins. Although this rate difference appears to be due to the lower essentiality of extracellular proteins than intracellular proteins in yeast, we here show that, in mammals, the impact of extracellularity is independent from the impact of gene essentiality. Our partial correlation analysis indicated that the impact of extracellularity on mammalian protein evolutionary rate is also independent from those of tissue-specificity, expression level, gene compactness, and the number of protein-protein interactions and, surprisingly, is the strongest among all the factors we examined. Similar results were also found from principal component regression analysis. Our findings suggest that different rules govern the pace of protein sequence evolution in mammals and yeasts.

A focus on extracellular Ca2+ entry into skeletal muscle

PubMed Central

Cho, Chung-Hyun; Woo, Jin Seok; Perez, Claudio F; Lee, Eun Hui

2017-01-01

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca2+ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca2+ level is mainly determined by Ca2+ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca2+ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca2+ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca2+ entryway into skeletal muscle. Herein, recent studies on extracellular Ca2+ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca2+ entry and their influences on skeletal muscle function and disease. PMID:28912570

Stimulus-Dependent Dopamine Release in Attention-Deficit/Hyperactivity Disorder

ERIC Educational Resources Information Center

Sikstrom, Sverker; Soderlund, Goran

2007-01-01

Attention-deficit/hyperactivity disorder (ADHD) is related to an attenuated and dysfunctional dopamine system. Normally, a high extracellular dopamine level yields a tonic dopaminergic input that down-regulates stimuli-evoked phasic dopamine responses through autoreceptors. Abnormally low tonic extracellular dopamine in ADHD up-regulates the…

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

PubMed Central

Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

2015-01-01

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID:26042044

Use of glucose biosensors to measure extracellular glucose exudation by intertidal microphytobenthos in southern Tasmania.

PubMed

McMinn, Andrew; Lee, Shihong

2018-06-01

Micro glucose biosensors were used to measure net extracellular glucose produced by natural microphytobenthos and three diatom cultures (Amphora coffeaeformis, Navicula menisculus, Nitzschia longissima) from southern Tasmania, Australia. They were exposed to a light gradient in either nutrient-replete or nutrient-limiting conditions. Glucose exudation in the natural communities increased with increased light but the response in the cultures was variable. Similarly, nutrient-replete conditions elicited lower rates of glucose exudation in the natural communities but produced variable species-specific responses in the cultures. Increased glucose exudation mostly correlated with a reduction in maximum quantum yield (F v /F m ). The same trend was observed in the natural communities for relative maximum electron transfer rates (rETR max ) but responses in the cultures were again variable and species-specific. Responses of the three species to increased light and nutrient deficiency were variable, although glucose exudation, F v /F m and rETR max was mostly lower in the nutrient-limited media. In a second set of experiments species/communities were treated with/without antibiotics. In the dark, glucose concentrations in treatments with antibiotics remained unchanged, while in those with bacteria, it fell rapidly. In the sediment communities, glucose consumption in the dark was ~25% the rate of exudation at the highest light level. In culture, exudation rates were up to 100% greater than those with active bacteria. Rates of glucose consumption in the dark in the antibiotic-treated samples were negligible and up to 10 4 times lower than those with active bacteria. These results demonstrate the important role extracellular glucose exudation has on maintaining an active microbial loop. © 2018 Phycological Society of America.

Recent exposure to ultrafine particles in school children alters miR-222 expression in the extracellular fraction of saliva.

PubMed

Vriens, Annette; Nawrot, Tim S; Saenen, Nelly D; Provost, Eline B; Kicinski, Michal; Lefebvre, Wouter; Vanpoucke, Charlotte; Van Deun, Jan; De Wever, Olivier; Vrijens, Karen; De Boever, Patrick; Plusquin, Michelle

2016-07-26

Ultrafine particles (<100 nm) are ubiquitous present in the air and may contribute to adverse cardiovascular effects. Exposure to air pollutants can alter miRNA expression, which can affect downstream signaling pathways. miRNAs are present both in the intracellular and extracellular environment. In adults, miR-222 and miR-146a were identified as associated with particulate matter exposure. However, there is little evidence of molecular effects of ambient air pollution in children. This study examined whether exposure to fine and ultrafine particulate matter (PM) is associated with changes in the extracellular content of miR-222 and miR-146a of children. Saliva was collected from 80 children at two different time points, circa 11 weeks apart and stabilized for RNA preservation. The extracellular fraction of saliva was obtained by means of differential centrifugation and ultracentrifugation. Expression levels of miR-222 and miR-146a were profiled by qPCR. We regressed the extracellular miRNA expression against recent exposure to ultrafine and fine particles measured at the school site using mixed models, while accounting for sex, age, BMI, passive smoking, maternal education, hours of television use, time of the day and day of the week. Exposure to ultrafine particles (UFP) at the school site was positively associated with miR-222 expression in the extracellular fraction in saliva. For each IQR increase in particles in the class room (+8504 particles/cm(3)) or playground (+28776 particles/cm(3)), miR-222 was, respectively 23.5 % (95 % CI: 3.5 %-41.1 %; p = 0.021) or 29.9 % (95 % CI:10.6 %-49.1 %; p = 0.0027) higher. No associations were found between miR-146a and recent exposure to fine and ultrafine particles. Our results suggest a possible epigenetic mechanism via which cells respond rapidly to small particles, as exemplified by miR-222 changes in the extracellular fraction of saliva.

Activation of the prelimbic medial prefrontal cortex induces anxiety-like behaviors via N-Methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in mice.

PubMed

Saitoh, Akiyoshi; Ohashi, Masanori; Suzuki, Satoshi; Tsukagoshi, Mai; Sugiyama, Azusa; Yamada, Misa; Oka, Jun-Ichiro; Inagaki, Masatoshi; Yamada, Mitsuhiko

2014-08-01

We investigated the possible roles of the prelimbic medial prefrontal cortex (PL) in the regulation of anxiety-like behaviors by pharmacologically activating the terminals of neuronal inputs or postsynaptic efferent neurons with a sodium channel activator veratrine. The extracellular glutamate levels were measured by in vivo microdialysis, and the behaviors were assessed with the open field (OF) test in mice simultaneously. The samples were collected every 10 min for 60 min, as basal levels of glutamate. The medium containing drugs were perfused for 30 min. The OF test was performed in the last 10 min of drug perfusion. After the drug treatments, the perfusion medium containing drugs was switched back to perfusion medium without drugs, and then samples were collected for another 90 min. The extracellular glutamate levels were significantly elevated after local perfusion of veratrine in the PL. At the same time, perfusion of veratrine in the PL produced anxiety-like behaviors in mice. Local coperfusion of a sodium channel blocker, lamotrigine, completely diminished the veratrine-induced elevated extracellular glutamate levels and the behavioral changes. Local coperfusion of an NMDA receptor antagonist, MK-801, but not a non-NMDA (AMPA/kainate) receptor antagonist, CNQX, completely diminished the behavioral changes without any effects on the veratrine-induced elevated extracellular glutamate levels. This study demonstrates that the activation of the PL with veratrine induces anxiety-like behaviors via NMDA receptor-mediated glutamatergic neurotransmission in mice. © 2014 Wiley Periodicals, Inc.

Concise Review: Developing Best‐Practice Models for the Therapeutic Use of Extracellular Vesicles

PubMed Central

Reiner, Agnes T.; Witwer, Kenneth W.; van Balkom, Bas W.M.; de Beer, Joel; Brodie, Chaya; Corteling, Randolph L.; Gabrielsson, Susanne; Gimona, Mario; Ibrahim, Ahmed G.; de Kleijn, Dominique; Lai, Charles P.; Lötvall, Jan; del Portillo, Hernando A.; Reischl, Ilona G.; Riazifar, Milad; Salomon, Carlos; Tahara, Hidetoshi; Toh, Wei Seong; Wauben, Marca H.M.; Yang, Vicky K.; Yang, Yijun; Yeo, Ronne Wee Yeh; Yin, Hang; Giebel, Bernd

2017-01-01

Abstract Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell‐related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV‐based therapeutics at the preclinical and clinical levels. This review outlines topic‐specific action items that, if addressed, will enhance the development of best‐practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730–1739 PMID:28714557

Improving the cytoplasmic maturation of bovine oocytes matured in vitro with intracellular and/or extracellular antioxidants is not associated with increased rates of embryo development.

PubMed

Rocha-Frigoni, Nathália A S; Leão, Beatriz C S; Dall'Acqua, Priscila Chediek; Mingoti, Gisele Z

2016-11-01

The production of reactive oxygen species (ROS) is a normal process that occurs in the cellular mitochondrial respiratory chain. However, an increase in ROS levels during in vitro production of bovine embryos induces oxidative stress, leading to failed embryonic development. Therefore, we investigated whether supplementation of IVM medium with intracellular (cysteine and cysteamine; C + C) and/or extracellular (catalase; CAT) antioxidants improves the culture system, affects the mitochondrial membrane potential, affects the intracellular levels of ROS and glutathione (GSH) in the bovine oocytes at the end of maturation, and thereby affects the subsequent embryonic development. At the end of IVM, the metaphase II rates were unaffected by the treatments (76.7 ± 1.7% to 80.6 ± 5.2%; P > 0.05). The intracellular ROS levels, expressed in arbitrary fluorescence units, found in the oocytes treated with intracellular antioxidants (C + C and C + C + CAT groups; 1.06, averaged) were as low as those observed in immature oocytes (0 hour: 1.00 ± 0.12). Among mature oocytes, higher (P < 0.05) ROS levels were found in the control group (1.91 ± 0.10) when compared to the ROS levels found in oocytes treated with antioxidants. Intracellular GSH levels in all groups were lower (0.17 ± 0.09 to 0.51 ± 0.05; P < 0.05) than those in immature oocytes (1.00 ± 0.08), although GSH levels in the C + C group (0.51 ± 0.05) were greater (P < 0.05) than in the control, CAT, and C + C + CAT groups (0.23; averaged). The mitochondrial membrane potential in all groups was improved (1.6; averaged; P < 0.05) compared to the membrane potential observed in the immature oocytes (1.00 ± 0.05), with the exception of the C + C group (0.94 ± 0.03). There was no effect (P > 0.05) of antioxidant supplementation on embryonic development to the blastocyst stage (36.1%; averaged); however, there was an increased tendency (P = 0.0689) to obtain a higher blastocyst rate for the C + C + CAT group (47.5 ± 5.6%) compared to the control group (29.9 ± 4.8%). In conclusion, despite improvements in specific parameters of cytoplasmic maturation, the addition of intracellular and/or extracellular antioxidants during IVM did not affect embryo development. Copyright © 2016 Elsevier Inc. All rights reserved.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

PubMed Central

2012-01-01

Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins. PMID:22234238

Verticillium longisporum Infection Affects the Leaf Apoplastic Proteome, Metabolome, and Cell Wall Properties in Arabidopsis thaliana

PubMed Central

Floerl, Saskia; Majcherczyk, Andrzej; Possienke, Mareike; Feussner, Kirstin; Tappe, Hella; Gatz, Christiane; Feussner, Ivo; Kües, Ursula; Polle, Andrea

2012-01-01

Verticillium longisporum (VL) is one of the most devastating diseases in important oil crops from the family of Brassicaceae. The fungus resides for much time of its life cycle in the extracellular fluid of the vascular system, where it cannot be controlled by conventional fungicides. To obtain insights into the biology of VL-plant interaction in the apoplast, the secretome consisting of the extracellular proteome and metabolome as well as cell wall properties were studied in the model Brassicaceae, Arabidopsis thaliana. VL infection resulted in increased production of cell wall material with an altered composition of carbohydrate polymers and increased lignification. The abundance of several hundred soluble metabolites changed in the apoplast of VL-infected plants including signalling and defence compounds such as glycosides of salicylic acid, lignans and dihydroxybenzoic acid as well as oxylipins. The extracellular proteome of healthy leaves was enriched in antifungal proteins. VL caused specific increases in six apoplast proteins (three peroxidases PRX52, PRX34, P37, serine carboxypeptidase SCPL20, α-galactosidase AGAL2 and a germin-like protein GLP3), which have functions in defence and cell wall modification. The abundance of a lectin-like, chitin-inducible protein (CILLP) was reduced. Since the transcript levels of most of the induced proteins were not elevated until late infection time points (>20 dpi), whereas those of CILLP and GLP3 were reduced at earlier time points, our results may suggest that VL enhances its virulence by rapid down-regulation and delay of induction of plant defence genes. PMID:22363647

Detection and localisation of the abalone probiotic Vibrio midae SY9 and its extracellular protease, VmproA, within the digestive tract of the South African abalone, Haliotis midae.

PubMed

Huddy, Robert J; Coyne, Vernon E

2014-01-01

Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone.

Oxygen-Inducible Glutamate Oxaloacetate Transaminase as Protective Switch Transforming Neurotoxic Glutamate to Metabolic Fuel During Acute Ischemic Stroke

PubMed Central

Rink, Cameron; Gnyawali, Surya; Peterson, Laura

2011-01-01

Abstract This work rests on our previous report (J Cereb Blood Flow Metab 30: 1275–1287, 2010) recognizing that glutamate (Glu) oxaloacetate transaminase (GOT) is induced when brain tissue hypoxia is corrected during acute ischemic stroke (AIS). GOT can metabolize Glu into tricarboxylic acid cycle intermediates and may therefore be useful to harness excess neurotoxic extracellular Glu during AIS as a metabolic substrate. We report that in cultured neural cells challenged with hypoglycemia, extracellular Glu can support cell survival as long as there is sufficient oxygenation. This effect is abrogated by GOT knockdown. In a rodent model of AIS, supplemental oxygen (100% O2 inhaled) during ischemia significantly increased GOT expression and activity in the stroke-affected brain tissue and prevented loss of ATP. Biochemical analyses and in vivo magnetic resonance spectroscopy during stroke demonstrated that such elevated GOT decreased Glu levels at the stroke-affected site. In vivo lentiviral gene delivery of GOT minimized lesion volume, whereas GOT knockdown worsened stroke outcomes. Thus, brain tissue GOT emerges as a novel target in managing stroke outcomes. This work demonstrates that correction of hypoxia during AIS can help clear extracellular neurotoxic Glu by enabling utilization of this amino acid as a metabolic fuel to support survival of the hypoglycemic brain tissue. Strategies to mitigate extracellular Glu-mediated neurodegeneration via blocking receptor-mediated excitotoxicity have failed in clinical trials. We introduce the concept that under hypoglycemic conditions extracellular Glu can be transformed from a neurotoxin to a survival factor by GOT, provided there is sufficient oxygen to sustain cellular respiration. Antioxid. Redox Signal. 14, 1777–1785. PMID:21361730

Extracellular forms of IL-37 inhibit innate inflammation in vitro and in vivo but require the IL-1 family decoy receptor IL-1R8.

PubMed

Li, Suzhao; Neff, C Preston; Barber, Kristina; Hong, Jaewoo; Luo, Yuchun; Azam, Tania; Palmer, Brent E; Fujita, Mayumi; Garlanda, Cecilia; Mantovani, Alberto; Kim, Soohyun; Dinarello, Charles Anthony

2015-02-24

Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1β (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture

NASA Astrophysics Data System (ADS)

Abbah, Sunny A.; Thomas, Dilip; Browne, Shane; O'Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

2016-02-01

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-β1). In tendon tissue repair, increased activity of TGF-β1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-β1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-β1 and a range of pro-fibrotic extracellular matrix genes.

Effects of coarse chalk dust particles (2.5-10 μm) on respiratory burst and oxidative stress in alveolar macrophages.

PubMed

Zhang, Yuexia; Yang, Zhenhua; Feng, Yan; Li, Ruijin; Zhang, Quanxi; Geng, Hong; Dong, Chuan

2015-08-01

The main aim of the present study was to examine in vitro responses of rat alveolar macrophages (AMs) exposed to coarse chalk dust particles (particulate matter in the size range 2.5-10 μm, PM(coarse)) by respiratory burst and oxidative stress. Chalk PM(coarse)-induced respiratory burst in AMs was measured by using a luminol-dependent chemiluminescence (CL) method. Also, the cell viability; lactate dehydrogenase (LDH) release; levels of cellular superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and acid phosphatase (ACP); plasma membrane ATPase; and extracellular nitric oxide (NO) level were determined 4 h following the treatment with the different dosages of chalk PM(coarse). The results showed that chalk PM(coarse) initiated the respiratory burst of AMs as indicated by strong CL, which was inhibited by diphenyleneiodonium chloride and L-N-nitro-L-arginine methyl ester hydrochloride. It suggested that chalk PM(coarse) induced the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in AMs. This hypothesis was confirmed by the fact that chalk PM(coarse) resulted in a significant decrease of intracellular SOD, GSH, ACP, and ATPase levels and a notable increase of intracellular CAT, MDA content, and extracellular NO level, consequently leading to a decrease of the cell viability and a increase of LDH release. It was concluded that AMs exposed to chalk PM(coarse) can suffer from cytotoxicity which may be mediated by generation of excessive ROS/RNS. Graphical Abstract The possible mechanism of coarse chalk particles-induced adverse effects in AMs.

Adhesion-Dependent Redistribution of MAP Kinase and MEK Promotes Muscarinic Receptor-Mediated Signaling to the Nucleus

PubMed Central

Slack, Barbara E.; Siniaia, Marina S.

2008-01-01

The mitogen-activated protein kinases (MAPKs) are activated by extracellular signals, and translocate to the nucleus where they modulate transcription. Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins is required for efficient transmission of MAPK-based signals initiated by growth factors. However, the modulation of G protein-coupled receptor (GPCR) signaling by adhesion is less well understood. In the present study we assessed the impact of cell adhesion on MAPK activation by muscarinic M3 receptors. The muscarinic agonist carbachol more efficiently promoted stress fiber formation and tyrosine phosphorylation of focal adhesion-associated proteins in M3 receptor-expressing cells adherent to fibronectin or collagen type I, as compared to polylysine. Overall MAPK activation was robust in cells adherent to all three substrata. However, total levels of MAPK and mitogen-activated protein kinase kinase (MEK) in the nucleus were significantly greater in cells adherent to ECM proteins for 2.5 hours, and levels of activated MAPK and MEK in the nuclei of these cells were higher following carbachol stimulation, relative to levels in cells adherent to polylysine. MEK inhibitors did not prevent adhesion-dependent translocation of MAPK and MEK to the nucleus, and increased nuclear phospho-MEK levels in carbachol-stimulated cells. The results suggest that adhesion of cells to ECM triggers the redistribution of MAPK and MEK to the nucleus, possibly as a result of the cytoskeletal rearrangements that accompany cell spreading. This may represent a mechanism for priming the nucleus with MEK and MAPK, leading to more rapid and pronounced increases in intranuclear phospho-MAPK upon GPCR stimulation. PMID:15779001

Brain intra- and extracellular sodium concentration in multiple sclerosis: a 7 T MRI study.

PubMed

Petracca, Maria; Vancea, Roxana O; Fleysher, Lazar; Jonkman, Laura E; Oesingmann, Niels; Inglese, Matilde

2016-03-01

Intra-axonal accumulation of sodium ions is one of the key mechanisms of delayed neuro-axonal degeneration that contributes to disability accrual in multiple sclerosis. In vivo sodium magnetic resonance imaging studies have demonstrated an increase of brain total sodium concentration in patients with multiple sclerosis, especially in patients with greater disability. However, total sodium concentration is a weighted average of intra- and extra-cellular sodium concentration whose changes reflect different tissue pathophysiological processes. The in vivo, non-invasive measurement of intracellular sodium concentration is quite challenging and the few applications in patients with neurological diseases are limited to case reports and qualitative assessments. In the present study we provide first evidence of the feasibility of triple quantum filtered (23)Na magnetic resonance imaging at 7 T, and provide in vivo quantification of global and regional brain intra- and extra-cellular sodium concentration in 19 relapsing-remitting multiple sclerosis patients and 17 heathy controls. Global grey matter and white matter total sodium concentration (respectively P < 0.05 and P < 0.01), and intracellular sodium concentration (both P < 0.001) were higher while grey matter and white matter intracellular sodium volume fraction (indirect measure of extracellular sodium concentration) were lower (respectively P = 0.62 and P < 0.001) in patients compared with healthy controls. At a brain regional level, clusters of increased total sodium concentration and intracellular sodium concentration and decreased intracellular sodium volume fraction were found in several cortical, subcortical and white matter regions when patients were compared with healthy controls (P < 0.05 family-wise error corrected for total sodium concentration, P < 0.05 uncorrected for multiple comparisons for intracellular sodium concentration and intracellular sodium volume fraction). Measures of total sodium concentration and intracellular sodium volume fraction, but not measures of intracellular sodium concentration were correlated with T2-weighted and T1-weighted lesion volumes (0.05 < P < 0.01) and with Expanded Disability Status Scale (P < 0.05). Thus, suggesting that while intracellular sodium volume fraction decrease could reflect expansion of extracellular space due to tissue loss, intracellular sodium concentration increase could reflect neuro-axonal metabolic dysfunction. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate.

PubMed

Belchí-Navarro, Sarai; Almagro, Lorena; Lijavetzky, Diego; Bru, Roque; Pedreño, María A

2012-01-01

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g(-1) dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g(-1) dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l(-1) sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l(-1), the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g(-1) dry weight g(-1) sucrose) using cyclodextrins and 25 μM methyljasmonate.

The administration of endocannabinoid uptake inhibitors OMDM-2 or VDM-11 promotes sleep and decreases extracellular levels of dopamine in rats.

PubMed

Murillo-Rodríguez, Eric; Palomero-Rivero, Marcela; Millán-Aldaco, Diana; Di Marzo, Vincenzo

2013-01-17

The family of the endocannabinoid system comprises endogenous lipids (such as anandamide [ANA]), receptors (CB(1)/CB(2) cannabinoid receptors), metabolic enzymes (fatty acid amide hydrolase [FAAH]) and a putative membrane transporter (anandamide membrane transporter [AMT]). Although the role of ANA, FAAH or the CB(1) cannabinoid receptor in sleep modulation has been reported, the effects of the inhibition of AMT on sleep remain unclear. In the present study, we show that microdialysis perfusion in rats of AMT inhibitors, (9Z)-N-[1-((R)-4-hydroxbenzyl)-2-hydroxyethyl]-9-octadecenamide (OMDM-2) or N-(4-hydroxy-2-methylphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (VDM-11; 10, 20 or 30 μM; each compound) delivered into the paraventricular thalamic nucleus (PVA) increased sleep and decreased waking. In addition, the infusion of compounds reduced the extracellular levels of dopamine collected from nucleus accumbens. Taken together, these findings illustrate a critical role of AMT in sleep modulation. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of acetylate hyperforin on the processing of amyloid precursor protein.

PubMed

Chen, Xiang; Feng, Wenshang; Chen, Qing; Yang, Xiangling; Yang, Depo; Wang, Dongmei; Zhong, Ling

2009-02-20

Hyperforin (HF) is a phloroglucinol compound obtained from St. John's Wort (Hypericum perforatum). Recent studies have shown that Hyperforin can be used to improve psychopathologic symptoms of Alzheimer's disease but the mechanism is not clear. This may be partly due to the difficult in studying Hyperforin, since this chemical is unstable and is sensitive to light, oxygen, and heat. In this study, we explored the effects of acetylate hyperforin (ace-HF), a stable derivative of hyperforin, on the processing of amyloid precursor protein (APP). HEK293 cells transfected with pcDNA3.1APP695sw and SH-SY5Y cells were treated with ace-HF, followed by measuring the levels of APP and sAPPα. Twelve hours of treatment led to an increase in extracellular sAPPα, but APP mRNA and protein levels were unchanged. Further studies with α-secretase and a pan PKC inhibitor, Calphostin C, indicated that ace-HF's effect on extracellular sAPPα was closely related to PKC activities and α-secretase activities. Our findings suggest that ace-HF can modulate α-secretase-mediated APP processing via a PKC signaling pathway.

Accumulation of Amyloid β-Protein in the Low-Density Membrane Domain Accurately Reflects the Extent of β-Amyloid Deposition in the Brain

PubMed Central

Oshima, Noriko; Morishima-Kawashima, Maho; Yamaguchi, Haruyasu; Yoshimura, Masahiro; Sugihara, Shiro; Khan, Karen; Games, Dora; Schenk, Dale; Ihara, Yasuo

2001-01-01

To learn more about the process of amyloid β-protein (Aβ) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Aβ content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Aβ. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Aβ deposits in those cases in which Aβ accumulated to significant levels. Aβ42 levels in the low-density membrane domain were proportional to the extent of total Aβ42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Aβ in a similar manner to aging humans, Aβ42 accumulation in the low-density membrane domain also increased as Aβ deposition progressed with aging. These observations indicate that the Aβ42 associated with low-density membrane domains is tightly coupled with the process of extracellular Aβ deposition. PMID:11395399

Targeting extracellular matrix remodeling in disease: Could resveratrol be a potential candidate?

PubMed

Agarwal, Renu; Agarwal, Puneet

2017-02-01

Disturbances of extracellular matrix homeostasis are associated with a number of pathological conditions. The ability of extracellular matrix to provide contextual information and hence control the individual or collective cellular behavior is increasingly being recognized. Hence, newer therapeutic approaches targeting extracellular matrix remodeling are widely investigated. We reviewed the current literature showing the effects of resveratrol on various aspects of extracellular matrix remodeling. This review presents a summary of the effects of resveratrol on extracellular matrix deposition and breakdown. Mechanisms of action of resveratrol in extracellular matrix deposition involving growth factors and their signaling pathways are discussed. Involvement of phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways and role of transcription factors and sirtuins on the effects of resveratrol on extracellular matrix homeostasis are summarized. It is evident from the literature presented in this review that resveratrol has significant effects on both the synthesis and breakdown of extracellular matrix. The major molecular targets of the action of resveratrol are growth factors and their signaling pathways, phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways, transcription factors, and SIRT-1. The effects of resveratrol on extracellular matrix and the molecular targets appear to be related to experimental models, experimental environment as well as the doses.

Targeting extracellular matrix remodeling in disease: Could resveratrol be a potential candidate?

PubMed Central

Agarwal, Puneet

2016-01-01

Disturbances of extracellular matrix homeostasis are associated with a number of pathological conditions. The ability of extracellular matrix to provide contextual information and hence control the individual or collective cellular behavior is increasingly being recognized. Hence, newer therapeutic approaches targeting extracellular matrix remodeling are widely investigated. We reviewed the current literature showing the effects of resveratrol on various aspects of extracellular matrix remodeling. This review presents a summary of the effects of resveratrol on extracellular matrix deposition and breakdown. Mechanisms of action of resveratrol in extracellular matrix deposition involving growth factors and their signaling pathways are discussed. Involvement of phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways and role of transcription factors and sirtuins on the effects of resveratrol on extracellular matrix homeostasis are summarized. It is evident from the literature presented in this review that resveratrol has significant effects on both the synthesis and breakdown of extracellular matrix. The major molecular targets of the action of resveratrol are growth factors and their signaling pathways, phosphoinositol-3-kinase/Akt and mitogen-activated protein kinase pathways, transcription factors, and SIRT-1. The effects of resveratrol on extracellular matrix and the molecular targets appear to be related to experimental models, experimental environment as well as the doses. PMID:27798117

Interplay of VEGFa and MMP2 regulates invasion of glioblastoma.

PubMed

Gong, Jie; Zhu, Shugan; Zhang, Yuan; Wang, Jiangang

2014-12-01

Neovascularization plays a substantial role in the regulation of invasion of glioblastoma. However, the underlying molecular basis remains largely unknown. Both vascular endothelial growth factor a (VEGFa) and matrix metalloproteinases 2 (MMP2) are essential for cancer neovascularization and cancer invasion in that they promote endothelial mitogenesis and permeability, and promote extracellular matrix degradation, respectively. In the current study, we found strong positive correlation of VEGFa and phosphorylated MMP2 levels in the glioblastoma from the patients. Thus, we used a human glioblastoma line, A-172, to examine the interaction of VEGFa and MMP2. We found that overexpression of VEGFa in A-172 cells increased MMP2 levels, while inhibition of VEGFa in A-172 cells decreased MMP2 levels. On the other hand, forced changes in MMP2 levels in A-172 cells did not affect VEGFa levels. These data suggest that VEGFa may regulate MMP2 in glioblastoma, while MMP2 did not appear to affect VEGFa levels. We then examined the signaling pathways involved in the regulation of MMP2 levels by VEGFa. Application of a specific extracellular-related kinase 1/2 (ERK1/2) inhibitor, but not application of either an protein kinase B (Akt) inhibitor, or a Jun N-terminal kinase (JNK) inhibitor to VEGFa-overexpressing A-172 cells substantially abolished the effect of VEGFa on MMP2 activation, suggesting that VEGFa may increase MMP2 levels via ERK/mitogen-activated protein kinase (MAPK), but not phosphatidylinositol 3-kinase (PI3K) or JNK signaling pathways in glioblastoma. Moreover, adapted VEGFa levels were found to directly and positively affect the glioblastoma development in an intracranial glioblastoma implantation model. Taken together, our data suggest that anti-VEGFa treatment in glioblastoma may inhibit neovascularization not only by VEGFa itself but also by its regulatory effect on MMP2.

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease.

PubMed

Li, Fan; Li, Lisha; Cheng, Meijuan; Wang, Xiumin; Hao, Jun; Liu, Shuxia; Duan, Huijun

2017-01-22

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD. Copyright © 2016 Elsevier Inc. All rights reserved.

The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

PubMed

Nocito, Laura; Kleckner, Amber S; Yoo, Elsia J; Jones Iv, Albert R; Liesa, Marc; Corkey, Barbara E

2015-01-01

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

The Extracellular Redox State Modulates Mitochondrial Function, Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

PubMed Central

Nocito, Laura; Kleckner, Amber S.; Yoo, Elsia J.; Jones IV, Albert R.; Liesa, Marc; Corkey, Barbara E.

2015-01-01

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases. PMID:25816337

Electrodiffusive Model for Astrocytic and Neuronal Ion Concentration Dynamics

PubMed Central

Halnes, Geir; Østby, Ivar; Pettersen, Klas H.; Omholt, Stig W.; Einevoll, Gaute T.

2013-01-01

The cable equation is a proper framework for modeling electrical neural signalling that takes place at a timescale at which the ionic concentrations vary little. However, in neural tissue there are also key dynamic processes that occur at longer timescales. For example, endured periods of intense neural signaling may cause the local extracellular K+-concentration to increase by several millimolars. The clearance of this excess K+ depends partly on diffusion in the extracellular space, partly on local uptake by astrocytes, and partly on intracellular transport (spatial buffering) within astrocytes. These processes, that take place at the time scale of seconds, demand a mathematical description able to account for the spatiotemporal variations in ion concentrations as well as the subsequent effects of these variations on the membrane potential. Here, we present a general electrodiffusive formalism for modeling of ion concentration dynamics in a one-dimensional geometry, including both the intra- and extracellular domains. Based on the Nernst-Planck equations, this formalism ensures that the membrane potential and ion concentrations are in consistency, it ensures global particle/charge conservation and it accounts for diffusion and concentration dependent variations in resistivity. We apply the formalism to a model of astrocytes exchanging ions with the extracellular space. The simulations show that K+-removal from high-concentration regions is driven by a local depolarization of the astrocyte membrane, which concertedly (i) increases the local astrocytic uptake of K+, (ii) suppresses extracellular transport of K+, (iii) increases axial transport of K+ within astrocytes, and (iv) facilitates astrocytic relase of K+ in regions where the extracellular concentration is low. Together, these mechanisms seem to provide a robust regulatory scheme for shielding the extracellular space from excess K+. PMID:24367247

Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions.

PubMed

Yegutkin, Gennady G; Guerrero-Toro, Cindy; Kilinc, Erkan; Koroleva, Kseniya; Ishchenko, Yevheniia; Abushik, Polina; Giniatullina, Raisa; Fayuk, Dmitriy; Giniatullin, Rashid

2016-09-01

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca(2+) transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.

Enhanced osteogenic differentiation of MC3T3-E1 cells on grid-topographic surface and evidence for involvement of YAP mediator.

PubMed

Zhang, Yingying; Gong, He; Sun, Yan; Huang, Yan; Fan, Yubo

2016-05-01

Numerous studies have shown that surface topography can promote cell-substrate associations and deeply influence cell fate. The intracellular mechanism or how micro- or nano-patterned extracellular signal is ultimately linked to activity of nuclear transcription factors remains unknown. It has been reported that Yes-associated protein (YAP) can respond to extracellular matrix microenvironment signals, thus regulates stem cell differentiation process. We propose that YAP may play a role in mediating the topography induced cell differentiation. To this end, we fabricated polydimethylsiloxane (PDMS) micropatterns with grid topology (GT) (3 μm pattern width, 2 μm pattern interval length, 7 μm pattern height); nonpatterned PDMS substrates were used as the planar controls. The MC3T3-E1 cells were then cultured on these surfaces, respectively, in osteogenic inducing medium. Cell differentiation in terms of osteogenesis related gene expression, protein levels, alkaline phosphatase activity and extracellular matrix mineralization was assessed. It was shown that the cells on GT surfaces had stronger osteogenesis capacity. In addition, expression level of YAP was increased when MC3T3-E1 cells grew on GT substrates, which was similar to the levels of osteogenic differentiation markers. It was also shown that YAP knockdown attenuated GT substrates-induced MC3T3-E1 differentiation, which reduced the osteogenic differentiation effect of the GT substrates. Collectively, our findings indicate that GT substrates-induced MC3T3-E1 differentiation may be associated with YAP. This paper provides new target points for transcriptional mechanism research of microenvironment induced cell differentiation and a useful approach to obtain more biofunctionalization scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc.

Apyrase (Nucleoside Triphosphate-Diphosphohydrolase) and Extracellular Nucleotides Regulate Cotton Fiber Elongation in Cultured Ovules1[W][OA

PubMed Central

Clark, Greg; Torres, Jonathan; Finlayson, Scott; Guan, Xueying; Handley, Craig; Lee, Jinsuk; Kays, Julia E.; Chen, Z. Jeffery; Roux, Stanley J.

2010-01-01

Ectoapyrase enzymes remove the terminal phosphate from extracellular nucleoside tri- and diphosphates. In Arabidopsis (Arabidopsis thaliana), two ectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulators of growth. In fibers of cotton (Gossypium hirsutum), transcript levels for GhAPY1 and GhAPY2, two closely related ectoapyrases that have high sequence similarity to AtAPY1 and AtAPY2, are up-regulated when fibers enter their rapid growth phase. In an ovule culture system, fibers release ATP as they grow, and when their ectoapyrase activity is blocked by the addition of polyclonal anti-apyrase antibodies or by two different small molecule inhibitors, the medium ATP level rises and fiber growth is suppressed. High concentrations of the poorly hydrolyzable nucleotides ATPγS and ADPβS applied to the medium inhibit fiber growth, and low concentrations of them stimulate growth, but treatment with adenosine 5′-O-thiomonophosphate causes no change in the growth rate. Both the inhibition and stimulation of growth by applied nucleotides can be blocked by an antagonist that blocks purinoceptors in animal cells, and by adenosine. Treatment of cotton ovule cultures with ATPγS induces increased levels of ethylene, and two ethylene antagonists, aminovinylglycine and silver nitrate, block both the growth stimulatory and growth inhibitory effects of applied nucleotides. In addition, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, lowers the concentration of nucleotide needed to promote fiber growth. These data indicate that ectoapyrases and extracellular nucleotides play a significant role in regulating cotton fiber growth and that ethylene is a likely downstream component of the signaling pathway. PMID:20018604

Nitric oxide facilitates active avoidance learning via enhancement of glutamate levels in the hippocampal dentate gyrus.

PubMed

Wang, Shi; Pan, De-Xi; Wang, Dan; Wan, Peng; Qiu, De-Lai; Jin, Qing-Hua

2014-09-01

The hippocampus is a key structure for learning and memory in mammals, and long-term potentiation (LTP) is an important cellular mechanism responsible for learning and memory. Despite a number of studies indicating that nitric oxide (NO) is involved in the formation and maintenance of LTP as a retrograde messenger, few studies have used neurotransmitter release as a visual indicator in awake animals to explore the role of NO in learning-dependent long-term enhancement of synaptic efficiency. Therefore, in the present study, the effects of l-NMMA (a NO synthase inhibitor) and SNP (a NO donor) on extracellular glutamate (Glu) concentrations and amplitudes of field excitatory postsynaptic potential (fEPSP) were measured in the hippocampal dentate gyrus (DG) region during the acquisition and extinction of active-avoidance behavior in freely-moving conscious rats. In the control group, the extracellular concentration of Glu in the DG was significantly increased during the acquisition of active-avoidance behavior and gradually returned to baseline levels following extinction training. In the experimental group, the change in Glu concentration was significantly reduced by local microinjection of l-NMMA, as was the acquisition of the active-avoidance behavior. In contrast, the change in Glu concentration was significantly enhanced by SNP, and the acquisition of the active-avoidance behavior was significantly accelerated. Furthermore, in all groups, the changes in extracellular Glu were accompanied by corresponding changes in fEPSP amplitude and active-avoidance behavior. Our results suggest that NO in the hippocampal DG facilitates active avoidance learning via enhancements of glutamate levels and synaptic efficiency in rats. Copyright © 2014 Elsevier B.V. All rights reserved.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

PubMed

Clark, Melinda E; He, Zhili; Redding, Alyssa M; Joachimiak, Marcin P; Keasling, Jay D; Zhou, Jizhong Z; Arkin, Adam P; Mukhopadhyay, Aindrila; Fields, Matthew W

2012-04-16

Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state

PubMed Central

2012-01-01

Background Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. Results The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Conclusions Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion. PMID:22507456

Response of brain oxygenation and metabolism to deep hypothermic circulatory arrest in newborn piglets: comparison of pH-stat and alpha-stat strategies.

PubMed

Markowitz, Scott D; Mendoza-Paredes, Alberto; Liu, Huiping; Pastuszko, Peter; Schultz, Steven P; Schears, Gregory J; Greeley, William J; Wilson, David F; Pastuszko, Anna

2007-07-01

To determine the effect of pH-stat as compared with alpha-stat management on brain oxygenation, level of striatal extracellular dopamine, phosphorylation, and levels of protein kinase B (Akt) and cyclic adenosine 3', 5'-monophosphate response element-binding protein (CREB), and levels of extracellular signal-regulated kinase (ERK)1/2, Bcl-2, and Bax in a piglet model of deep hypothermic circulatory arrest (DHCA). The piglets were placed on cardiopulmonary bypass (CPB), cooled with pH-stat or alpha-stat to 18 degrees C, subjected to 90 minutes of DHCA, rewarmed, weaned from CPB, and maintained for two hours recovery. The cortical oxygen was measured by: quenching of phosphorescence; dopamine by microdialysis; phosphorylation of CREB (p-CREB), ERK (p-ERK) 1/2, Akt (p-Akt), and level of Bcl-2, Bax by Western blots. Oxygen pressure histograms for the microvasculature of the cortex show substantially higher oxygen levels during cooling and during the oxygen depletion period after cardiac arrest (up to 15 minutes) when using pH-stat compared with alpha-stat management. Significant increases in dopamine occurred at 45 minutes and 60 minutes of DHCA in the alpha-stat and pH-stat groups, respectively. The p-CREB and p-Akt in the pH-stat group were significantly higher than in the alpha-stat group (140 +/- 9%, p < 0.05 and 125 +/- 6%, p < 0.05, respectively). There was no significant difference in p-ERK1/2 and Bax. The Bcl-2 increased in the pH-stat group to 121 +/- 4% (p < 0.05) compared with the alpha-stat group. The ratio Bcl-2:Bax increased in the pH-stat group compared with the alpha-stat group. The increase in p-CREB, p-Akt, Bcl-2, Bcl-2/Bax, and delay in increase of dopamine indicated that pH-stat, in the piglet model, prolongs "safe" time of DHCA and provides some brain protection against ischemic injury.

Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma☆

PubMed Central

Norian, John M.; Owen, Carter M.; Taboas, Juan; Korecki, Casey; Tuan, Rocky; Malik, Minnie; Catherino, William H.; Segars, James H.

2012-01-01

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor’s inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells. PMID:21983114

Curcumin analog EF24 induces apoptosis and downregulates the mitogen activated protein kinase/extracellular signal-regulated signaling pathway in oral squamous cell carcinoma.

PubMed

Lin, Chongxiang; Tu, Chengwei; Ma, Yike; Ye, Pengcheng; Shao, Xia; Yang, Zhaoan; Fang, Yiming

2017-10-01

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Diphenyldifluoroketone (EF24) is a curcumin analog that has been demonstrated to improve anticancer activity; however, its therapeutic potential and mechanisms in oral cancer remain unknown. In the present study, the effect of EF24 on apoptosis induction and its potential underlying mechanism in the CAL‑27 human OSCC cell line was investigated. To achieve this, various concentrations of cisplatin or EF24 were administrated to CAL‑27 cells for 24 h, and cell viability, apoptotic DNA fragmentation, and cleaved caspase 3 and 9 levels were evaluated. To investigate the potential underlying mechanism, the levels of mitogen‑activated protein kinase kinase 1 (MEK1) and extracellular signal‑regulated kinase (ERK), two key proteins in the mitogen‑activated protein kinase/ERK signaling pathway, were additionally examined. The results indicated that EF24 and cisplatin treatment decreased cell viability. EF24 treatment increased the levels of activated caspase 3 and 9, and decreased the phosphorylated forms of MEK1 and ERK. Sequential treatments of EF24 and 12‑phorbol‑13‑myristate acetate, a MAPK/ERK activator, resulted in a significant increase of activated MEK1 and ERK, and reversed cell viability. These results suggested that EF24 has potent anti‑tumor activity in OSCC via deactivation of the MAPK/ERK signaling pathway. Further analyses using animal models are required to confirm these findings in vivo.

Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance.

PubMed

Hayata, Hiroki; Miyazaki, Hiroaki; Niisato, Naomi; Yokoyama, Noriko; Marunaka, Yoshinori

2014-02-28

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells. Copyright © 2014. Published by Elsevier Inc.

Extracellular proteome analysis of Leptospira interrogans serovar Lai.

PubMed

Zeng, Lingbing; Zhang, Yunyi; Zhu, Yongzhang; Yin, Haidi; Zhuang, Xuran; Zhu, Weinan; Guo, Xiaokui; Qin, Jinhong

2013-10-01

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

Extracellular and Intracellular Cyclophilin A, Native and Post-Translationally Modified, Show Diverse and Specific Pathological Roles in Diseases.

PubMed

Xue, Chao; Sowden, Mark P; Berk, Bradford C

2018-05-01

CypA (cyclophilin A) is a ubiquitous and highly conserved protein with peptidyl prolyl isomerase activity. Because of its highly abundant level in the cytoplasm, most studies have focused on the roles of CypA as an intracellular protein. However, emerging evidence suggests an important role for extracellular CypA in the pathogenesis of several diseases through receptor (CD147 or other)-mediated autocrine and paracrine signaling pathways. In this review, we will discuss the shared and unique pathological roles of extracellular and intracellular CypA in human cardiovascular diseases. In addition, the evolving role of post-translational modifications of CypA in the pathogenesis of disease is discussed. Finally, recent studies with drugs specific for extracellular CypA show its importance in disease pathogenesis in several animal models and make extracellular CypA a new therapeutic target. © 2018 American Heart Association, Inc.

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

PubMed

Trigo, Gabriela; Ferreira, Paula; Ribeiro, Niza; Dinis, Márcia; Andrade, Elva Bonifácio; Melo-Cristino, José; Ramirez, Mário; Tavares, Delfina

2008-11-01

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.

Extracellular Zn2+ Influx into Nigral Dopaminergic Neurons Plays a Key Role for Pathogenesis of 6-Hydroxydopamine-Induced Parkinson's Disease in Rats.

PubMed

Tamano, Haruna; Nishio, Ryusuke; Morioka, Hiroki; Takeda, Atsushi

2018-04-29

Parkinson's disease (PD) is a progressive neurological disease characterized by a selective loss of nigrostriatal dopaminergic neurons. The exact cause of the neuronal loss remains unclear. Here, we report a unique mechanism of nigrostriatal dopaminergic neurodegeneration, in which extracellular Zn 2+ influx plays a key role for PD pathogenesis induced with 6-hydroxydopamine (6-OHDA) in rats. 6-OHDA rapidly increased intracellular Zn 2+ only in the substantia nigra pars compacta (SNpc) of brain slices and this increase was blocked in the presence of CaEDTA, an extracellular Zn 2+ chelator, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, indicating that 6-OHDA rapidly increases extracellular Zn 2+ influx via AMPA receptor activation in the SNpc. Extracellular Zn 2+ concentration was decreased under in vivo SNpc perfusion with 6-OHDA and this decrease was blocked by co-perfusion with CNQX, supporting 6-OHDA-induced Zn 2+ influx via AMPA receptor activation in the SNpc. Interestingly, both 6-OHDA-induced loss of nigrostriatal dopaminergic neurons and turning behavior to apomorphine were ameliorated by co-injection of intracellular Zn 2+ chelators, i.e., ZnAF-2DA and N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Co-injection of TPEN into the SNpc blocked 6-OHDA-induced increase in intracellular Zn 2+ but not in intracellular Ca 2+ . These results suggest that the rapid influx of extracellular Zn 2+ into dopaminergic neurons via AMPA receptor activation in the SNpc induces nigrostriatal dopaminergic neurodegeneration, resulting in 6-OHDA-induced PD in rats.

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm

PubMed Central

Stuart, Rhona K.; Mayali, Xavier; Boaro, Amy A.; Zemla, Adam; Everroad, R. Craig; Nilson, Daniel; Weber, Peter K.; Lipton, Mary; Bebout, Brad M.; Pett-Ridge, Jennifer

2016-01-01

ABSTRACT Although it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of 13C-labeled and 15N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively, of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM. PMID:27353754

Effects of VMAT2 inhibitors lobeline and GZ-793A on methamphetamine-induced changes in dopamine release, metabolism and synthesis in vivo.

PubMed

Meyer, Andrew C; Neugebauer, Nichole M; Zheng, Guangrong; Crooks, Peter A; Dwoskin, Linda P; Bardo, Michael T

2013-10-01

Vesicular monoamine transporter-2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N-(1,2R-dihydroxylpropyl)-2,6-cis-di(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A; 15 or 30 mg/kg) on METH-induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward-relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ-793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time-dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ-793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ-793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH-induced increase in extracellular DA. Both LOB and GZ-793A enhanced the duration of the METH-induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ-793A decreased synthesis; no effect of METH or GZ-793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time-dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ-793A to decrease METH reward. © 2013 International Society for Neurochemistry.

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm

DOE PAGES

Stuart, Rhona K.; Mayali, Xavier; Boaro, Amy A.; ...

2016-06-28

Here it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of 13C-labeled and 15N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively,more » of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM.« less

MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and NMDA receptors

PubMed Central

Collins, Stuart A.; Gudelsky, Gary A.; Yamamoto, Bryan K.

2015-01-01

MDMA is a widely abused psychostimulant which causes a rapid and robust release of the monoaminergic neurotransmitters dopamine and serotonin. Recently, it was shown that MDMA increases extracellular glutamate concentrations in the dorsal hippocampus, which is dependent on serotonin release and 5HT2A/2C receptor activation. The increased extracellular glutamate concentration coincides with a loss of parvalbumin-immunoreactive (PV-IR) interneurons of the dentate gyrus region. Given the known susceptibility of PV interneurons to excitotoxicity, we examined whether MDMA-induced increases in extracellular glutamate in the dentate gyrus are necessary for the loss of PV cells in rats. Extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Administration of the NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. Local administration of MDL100907, a selective 5HT2A receptor antagonist, prevented the increases in glutamate caused by reverse dialysis of MDMA directly into the dentate gyrus and prevented the reduction of PV-IR. These findings provide evidence that MDMA causes decreases in PV within the dentate gyrus through a 5HT2A receptor-mediated increase in glutamate and subsequent NMDA receptor activation. PMID:25936514

Extracellular ATP mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury.

PubMed

Shah, Dilip; Romero, Freddy; Stafstrom, William; Duong, Michelle; Summer, Ross

2014-01-01

Acute lung injury (ALI) is a severe inflammatory condition whose pathogenesis is irrevocably linked to neutrophil emigration to the lung. Activation and recruitment of neutrophils to the lung is mostly attributable to local production of the chemokines. However, much of our understanding of neutrophil recruitment to the lung is based on studies focusing on early time points after initiation of injury. In this study, we sought to evaluate the extended temporal relationship between neutrophil chemotactic factor expression and influx of neutrophils into the lung after intratracheal administration of either LPS or bleomycin. In both models, results demonstrated two phases of neutrophil chemotactic factor expression; first, an early phase characterized by high levels of CXCL1/keratinocyte-derived chemokine, CXCL2/monocyte-inhibitory protein-2, and CXCL5/LPS-induced chemokine expression, and second, a late phase distinguished by increases in extracellular ATP. Furthermore, we show that strategies aimed at either enhancing ATP catabolism (ip ecto-5'-nucleotidase administration) or inhibiting glycolytic ATP production (ip 2-deoxy-d-glucose treatment) reduce extracellular ATP accumulation, limit vascular leakage, and effectively block the late, but not the early, stages of neutrophil recruitment to the lung after LPS instillation. In conclusion, this study illustrates that neutrophil recruitment to the lung is mediated by the time-dependent expression of chemotactic factors and suggests that novel strategies, which reduce extracellular ATP accumulation, may attenuate late neutrophil recruitment and limit lung injury during ALI.

Extracellular ATP mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury

PubMed Central

Shah, Dilip; Romero, Freddy; Stafstrom, William; Duong, Michelle

2013-01-01

Acute lung injury (ALI) is a severe inflammatory condition whose pathogenesis is irrevocably linked to neutrophil emigration to the lung. Activation and recruitment of neutrophils to the lung is mostly attributable to local production of the chemokines. However, much of our understanding of neutrophil recruitment to the lung is based on studies focusing on early time points after initiation of injury. In this study, we sought to evaluate the extended temporal relationship between neutrophil chemotactic factor expression and influx of neutrophils into the lung after intratracheal administration of either LPS or bleomycin. In both models, results demonstrated two phases of neutrophil chemotactic factor expression; first, an early phase characterized by high levels of CXCL1/keratinocyte-derived chemokine, CXCL2/monocyte-inhibitory protein-2, and CXCL5/LPS-induced chemokine expression, and second, a late phase distinguished by increases in extracellular ATP. Furthermore, we show that strategies aimed at either enhancing ATP catabolism (ip ecto-5′-nucleotidase administration) or inhibiting glycolytic ATP production (ip 2-deoxy-d-glucose treatment) reduce extracellular ATP accumulation, limit vascular leakage, and effectively block the late, but not the early, stages of neutrophil recruitment to the lung after LPS instillation. In conclusion, this study illustrates that neutrophil recruitment to the lung is mediated by the time-dependent expression of chemotactic factors and suggests that novel strategies, which reduce extracellular ATP accumulation, may attenuate late neutrophil recruitment and limit lung injury during ALI. PMID:24285266

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

PubMed

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

High levels of circulating extracellular vesicles with altered expression and function during pregnancy.

PubMed

Nardi, Fabiola da Silva; Michelon, Tatiana Ferreira; Neumann, Jorge; Manvailer, Luis Felipe Santos; Wagner, Bettina; Horn, Peter A; Bicalho, Maria da Graça; Rebmann, Vera

2016-07-01

Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication and may interact with target cells locally and on a systemic level. Several studies had shown that circulating EVs' levels are increased during pregnancy. However, EVs characteristics, composition and biological functions in pregnancy still need to be clarified. This study aims to determine if circulating EVs during pregnancy are modified regarding levels, markers and cytokine profile as well as their reactivity towards peripheral blood cells. 26 pregnant women (PW) being in the second gestational trimester and 59 non-pregnant women (NPW) were investigated. EVs enrichment was performed by ExoQuick™ or ultracentrifugation; nanoparticle tracking analysis, SDS-PAGE followed by Western Blotting and densitometry, and IFN-γ, IL-10 and TGF-β1 ELISA for EVs characterization; imaging flow cytometry to analyze EVs' uptake by peripheral blood cells and flow cytometry were performed to analyze EVs function regarding induction of caspase-3 activity. Circulating EVs' levels were increased during pregnancy [26.9×10(6)EVs/ml (range: 6.4-46.3); p=0.003] vs NPW [18.9×10(6)EVs/ml (range: 2.5-61.3)]. Importantly, the immunosuppressive TGF-β1 and IL-10 cytokine cargo were increased in EVs of PW even after normalization to 1 million EVs [TGF-β1: 0.25pg/10(6)EVs (range: 0.0-2.0); p<0.0001] and [IL-10: 0.21pg/10(6)EVs (range: 0.0-16.8); p=0.006] vs NPW. Although EVs derived from non-pregnant and pregnant women were taken up by NK cells, the latter exclusively enhanced the caspase-3 activity in CD56(dim) NK cells (8.2±0.9; p=0.02). The qualitative and quantitative pregnancy-related alterations of circulating EVs provide first hints for an immune modulating role of circulating EVs during pregnancy. Copyright © 2016 Elsevier GmbH. All rights reserved.

Tangeretin triggers melanogenesis through the activation of melanogenic signaling proteins and sustained extracellular signal- regulated kinase in B16/F10 murine melanoma cells.

PubMed

Yoon, Hoon Seok; Ko, Hee-Chul; Kim, Sang Suk; Park, Kyung Jin; An, Hyun Joo; Choi, Young Hun; Kim, Se-Jae; Lee, Nam-Ho; Hyun, Chang-Gu

2015-03-01

In order to test the effectiveness of tangeretin at ameliorating melanoma and melanoma-associated depigmentation, western blotting was used to assess the melanin content of treated melanoma cells. Tangeretin, a 4',5,6,7,8-pentamethoxyflavone, was found to trigger intracellular melanin production in a concentration-dependent manner in B16/F10 murine melanoma cells. Melanin content increased 1.74-fold in response to treatment with 25 μM of tangeretin, compared to that in non-treated cells. Examination of melanogenic protein expression showed that tyrosinase, tyrosinase-related protein (TRP)-1, and extracellular signal-regulated kinase (ERK) 1/2 levels increased in a dose-dependent manner. Furthermore, the expression of cyclic adenosine monophosphate response element binding protein (CREB) and microphthalmia transcription factor (MITF) was increased by tangeretin in 1 h and 4 h, respectively. Tangeretin- upregulated melanogenesis was suppressed by ERK 1/2 inhibitor and not by ERK1 inhibitor. These results suggest that tangeretin has therapeutic potential for melanoma and melanoma-associated depigmentation because it can induce hyperpigmentation through the activation of melanogenic signaling proteins and initiation of sustained ERK2 expression.

Extracellular DNA Impedes the Transport of Vancomycin in Staphylococcus epidermidis Biofilms Preexposed to Subinhibitory Concentrations of Vancomycin

PubMed Central

Tseng, Boo Shan; Howlin, Robert P.; Deacon, Jill; Wharton, Julian A.; Thurner, Philipp J.; Gilmore, Brendan F.; Parsek, Matthew R.; Stoodley, Paul

2014-01-01

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular d-Ala-d-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections. PMID:25267673

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

PubMed Central

Lee, Mark J.; Liu, Hong; Barker, Bridget M.; Snarr, Brendan D.; Gravelat, Fabrice N.; Al Abdallah, Qusai; Gavino, Christina; Baistrocchi, Shane R.; Ostapska, Hanna; Xiao, Tianli; Ralph, Benjamin; Solis, Norma V.; Lehoux, Mélanie; Baptista, Stefanie D.; Thammahong, Arsa; Cerone, Robert P.; Kaminskyj, Susan G. W.; Guiot, Marie-Christine; Latgé, Jean-Paul; Fontaine, Thierry; Vinh, Donald C.; Filler, Scott G.; Sheppard, Donald C.

2015-01-01

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs. PMID:26492565

Aβ mediates Sigma receptor degradation via CaN/NFAT pathway

PubMed Central

Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

2016-01-01

Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation. PMID:27648137

Extracellular hyperosmolality and body temperature during physical exercise in dogs

NASA Technical Reports Server (NTRS)

Kozlowski, S.; Greenleaf, J. E.; Turlejska, E.; Nazar, K.

1980-01-01

The purpose of this study was to test the hypothesis that thermoregulation during exercise can be affected by extracellular fluid hyperosmolality without changing the plasma Na(+) concentration. The effects of preexercise venous infusions of hypertonic mannitol and NaCl solutions on rectal temperature responses were compared in dogs running at moderate intensity for 60 min on a treadmill. Plasma Na(+) concentration was increased by 12 meq after NaCl infusion, and decreased by 9 meq after mannitol infusion. Both infusions increased plasma by 15 mosmol/kg. After both infusions, rectal temperature was essentially constant during 60 min rest. However, compared with the noninfusion exercise increase in osmolality of 1.3 C, rectal temperature increased by 1.9 C after both postinfusion exercise experiments. It was concluded that inducing extracellular hyperosmolality, without elevating plasma, can induce excessive increases in rectal temperature during exericse but not at rest.

Low-level ultrahigh-frequency and ultrashort-pulse blue laser irradiation enhances osteoblast extracellular calcification by upregulating proliferation and differentiation via transient receptor potential vanilloid 1.

PubMed

Mikami, Risako; Mizutani, Koji; Aoki, Akira; Tamura, Yukihiko; Aoki, Kazuhiro; Izumi, Yuichi

2018-04-01

Low-level laser irradiation (LLLI) exerts various biostimulative effects, including promotion of wound healing and bone formation; however, few studies have examined biostimulation using blue lasers. The purpose of this study was to investigate the effects of low-level ultrahigh-frequency (UHF) and ultrashort-pulse (USP) blue laser irradiation on osteoblasts. The MC3T3-E1 osteoblast cell line was used in this study. Following LLLI with a 405 nm newly developed UHF-USP blue laser (80 MHz, 100 fs), osteoblast proliferation, and alkaline phosphatase (ALP) activity were assessed. In addition, mRNA levels of the osteoblast differentiation markers, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), and osteopontin (Opn) was evaluated, and extracellular calcification was quantified. To clarify the involvement of transient receptor potential (TRP) channels in LLLI-induced biostimulation, cells were treated prior to LLLI with capsazepine (CPZ), a selective inhibitor of TRP vanilloid 1 (TRPV1), and subsequent proliferation and ALP activity were measured. LLLI with the 405 nm UHF-USP blue laser significantly enhanced cell proliferation and ALP activity, compared with the non-irradiated control and LLLI using continuous-wave mode, without significant temperature elevation. LLLI promoted osteoblast proliferation in a dose-dependent manner up to 9.4 J/cm 2 and significantly accelerated cell proliferation in in vitro wound healing assay. ALP activity was significantly enhanced at doses up to 5.6 J/cm 2 , and expression of Osx and Alp mRNAs was significantly increased compared to that of the control on days 3 and 7 following LLLI at 5.6 J/cm 2 . The extent of extracellular calcification was also significantly higher as a result of LLLI 3 weeks after the treatment. Measurement of TRPV1 protein expression on 0, 3, and 7 days post-irradiation revealed no differences between the LLLI and control groups; however, promotion of cell proliferation and ALP activity by LLLI was significantly inhibited by CPZ. LLLI with a 405 nm UHF-USP blue laser enhances extracellular calcification of osteoblasts by upregulating proliferation and differentiation via TRPV1. Lasers Surg. Med. 50:340-352, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

PubMed Central

Riquelme, Manuel A.; Cea, Luis A.; Vega, José L.; Puebla, Carlos; Vargas, Aníbal A.; Shoji, Kenji F.; Subiabre, Mario; Sáez, Juan C.

2015-01-01

The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs. PMID:26000275

Time-Dependent Compensatory Responses to Chronic Neuroinflammation in Hippocampus and Brainstem: The Potential Role of Glutamate Neurotransmission

PubMed Central

Brothers, Holly M.; Bardou, Isabelle; Hopp, Sarah C.; Marchalant, Yannick; Kaercher, Roxanne M.; Turner, Sarah M.; Mitchem, Mollie R.; Kigerl, Kristina; Wenk, Gary L.

2014-01-01

Chronic neuroinflammation is characteristic of neurodegenerative diseases and is present during very early stages, yet significant pathology and behavioral deficits do not manifest until advanced age. We investigated the consequences of experimentally-induced chronic neuroinflammation within the hippocampus and brainstem of young (4 mo) F-344 rats. Lipopolysaccharide (LPS) was infused continuously into the IVth ventricle for 2, 4 or 8 weeks. The number of MHC II immunoreactive microglia in the brain continued to increase throughout the infusion period. In contrast, performance in the Morris water maze was impaired after 4 weeks but recovered by 8 weeks. Likewise, a transient loss of tyrosine hydroxylase immunoreactivity in the substantia nigra and locus coeruleus was observed after 2 weeks, but returned to control levels by 4 weeks of continuous LPS infusion. These data suggest that direct activation of microglia is sufficient to drive, but not sustain, spatial memory impairment and a decrease in tyrosine hydroxylase production in young rats. Our previous studies suggest that chronic neuroinflammation elevates extracellular glutamate and that this elevation underlies the spatial memory impairment. In the current study, increased levels of GLT1 and SNAP25 in the hippocampus corresponded with the resolution of performance deficit. Increased expression of SNAP25 is consistent with reduced glutamate release from axonal terminals while increased GLT1 is consistent with enhanced clearance of extracellular glutamate. These data demonstrate the capacity of the brain to compensate for the presence of chronic neuroinflammation, despite continued activation of microglia, through changes in the regulation of the glutamatergic system. PMID:24600537

The Role of Glutamate Release on Voltage-Dependent Anion Channels (VDAC)-Mediated Apoptosis in an Eleven Vessel Occlusion Model in Rats

PubMed Central

Park, Eunkuk; Lee, Gi-Ja; Choi, Samjin; Choi, Seok-Keun; Chae, Su-Jin; Kang, Sung-Wook; Pak, Youngmi Kim; Park, Hun-Kuk

2010-01-01

Voltage-dependent anion channel (VDAC) is the main protein in mitochondria-mediated apoptosis, and the modulation of VDAC may be induced by the excessive release of extracellular glutamate. This study examined the role of glutamate release on VDAC-mediated apoptosis in an eleven vessel occlusion model in rats. Male Sprague-Dawley rats (250–350 g) were used for the 11 vessel occlusion ischemic model, which were induced for a 10-min transient occlusion. During the ischemic and initial reperfusion episode, the real-time monitoring of the extracellular glutamate concentration was measured using an amperometric microdialysis biosensor and the cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry. To confirm neuronal apoptosis, the brains were removed 72 h after ischemia to detect the neuron-specific nuclear protein and pro-apoptotic proteins (cleaved caspase-3, VDAC, p53 and BAX). The changes in the mitochondrial morphology were measured by atomic force microscopy. A decrease in the % of CBF was observed, and an increase in glutamate release was detected after the onset of ischemia, which continued to increase during the ischemic period. A significantly higher level of glutamate release was observed in the ischemia group. The increased glutamate levels in the ischemia group resulted in the activation of VDAC and pro-apoptotic proteins in the hippocampus with morphological alterations to the mitochondria. This study suggests that an increase in glutamate release promotes VDAC-mediated apoptosis in an 11 vessel occlusion ischemic model. PMID:21203570

Identification of Molecular and Cellular Responses of Desulfovibrio vulgaris Biofilms under Culture Conditions Relevant to Field Conditions for Bioreduction of Toxic Metals and Radionuclides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judy D. Wall

2011-06-09

Our findings demonstrated that D. vulgaris surface-adhered populations produce extracellular structures, and that that the cells have altered carbon and energy flux compared to planktonic cells. Biofilms did not have greatly increased carbohydrate accumulation. Interestingly genes present on the native plasmid found in D. vulgaris Hildenborough were necessary for wild type biofilm formation. In addition, extracellular appendages dependent on functions or proteins encoded by flaG or fliA also contributed to biofilm formation. Studies with SRB biofilms have indicated that the reduction and precipitation of metals can occur within the biofilm matrix; however, little work has been done to elucidate themore » physiological state of surface-adhered cells during metal reduction (Cr6+, U6+) and how this process is affected by nutrient feed levels (i.e., the stimulant).« less

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system.

PubMed

Kajino, T; Saito, Y; Asami, O; Yamada, Y; Hirai, M; Udata, S

1997-10-01

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L-1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

PubMed

Souffriau, Ben; den Abt, Tom; Thevelein, Johan M

2012-07-30

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

PubMed Central

Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

2015-01-01

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

Biologic response of human anterior cruciate ligamentocytes on collagen-patches to platelet-rich plasma formulations with and without leucocytes.

PubMed

Krismer, Anna M; Cabra, Romina S; May, Rahel D; Frauchiger, Daniela A; Kohl, Sandro; Ahmad, Sufian S; Gantenbein, Benjamin

2017-12-01

Due to the poor self-healing capacities of the anterior cruciate ligament, previous primary repair attempts have failed. To enhance biologic healing, platelet rich plasma and collagen scaffold have shown promise in animal models. Platelet rich plasma (PRP) is already used in several clinical applications although outcomes are quite debated. The purpose of this study was to examine the effects of different PRP formulations during 21 days: With leucocytes and pure PRP on human anterior cruciate ligament-derived ligamentocytes grown on collagen patches in 3D cell cultures in vitro. Three experimental groups were formed: 2.5% leucocyte rich PRP, 2.5% pure PRP, 20% leucocyte rich PRP, a negative control, and a positive control. Cell proliferation, cell phenotype on mRNA transcript level, and extracellular matrix production (total collagen and glycosaminoglycan content) were evaluated. DNA content and metabolic cell activity increased significantly in all groups on day 21 compared to day 7, except in the negative control. No changes in extracellular matrix production were detected. Different catabolic genes were induced depending on the concentration of leucocyte rich PRP. PRP with and without leucocytes treated anterior cruciate ligamentocytes significantly increased cell proliferation but not extracellular matrix production. However, the specific activation of different catabolic genes was dependent on the relative content of leucocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2733-2739, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.